BIO 115 Lab Manual Spring 2023
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School
St. Joseph's College New York *
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Course
115
Subject
Biology
Date
Jan 9, 2024
Type
Pages
82
Uploaded by Smeezyy07
Introduction
to
Human
Inheritance
Bio
115
Biology
Department
St.
Joseph’'s
College
Lab
#1
BIOLOGY
115
Laboratory
1
USE
OF
THE
MICROSCOPE
Units
of
measurement
in
the
metric
system
All
measurernents
in
the
laboratory
are
in
the
meric
system,
To
express
the
size
of
small
objects,
such
as
cell
contenis,
biologists
use
even
smaller
units
of
the
metric
system.
‘
These
units
are
one
thousandth
of
each
other:
1
meter
=
100
centimeters
=
1000
millimeters
1
centimeter
=
10
millirneters
1
millimeter
=
1000
micromerers
1
micrometer
=
1000
nanometers
1
nanometer
=
1000
picomerers
To
demonstrate
that
you
understand
the
relationship
of
one
metric
unit
to
another
fill
in
the
blanks
below:
¢
1.5mm
=
micrometers
0.25
mm
=
micromerers
55
nm
=
e
mm
1.5¢m
=
:
mm
=
micromeaters
Cogx
[
.
Measure
the
diameter
of
this
circle
1o
the
nearest
millimerer:
The
circle
18
mim;
S
-
micrometers;
.
__nm
-
4
¢
Different
types
of
microscopes
and
their
uses
Lighr
Microscope
.
Light
microscopes
use
light
rays
thet
are
magnified
and
focused
by
means
of
lenses.
The
dissecting
microscope
Designed
to
study
entre
objects
in
three
dimension
at
low
magnification
The
compound
light
microscope
Used
for
exarnination
of
small
or
thinly
sliced
cross
or
longiwdinal
sections
of
objects
under
magni-
fication
that
is
higher
than
the
dissecring
microscope’s.
lllumination
is
from
below,
and
the
light
passes
through
clear
sections
but
does
not
pass
through
opaque
sections.
To
improve
conmast,
stains
or
dyes
are
used
that
bind
to
cellular
structures
and
absorb
light.
1/4
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Phase
contrast
microscopes
Used
for
observadon
of
living
ceils
and
tissue
since
the
light
out
of
phase
creates
contrast
between
different
cellular
organelles,
4
Nomarski
optics
|
|
variation
in
phase
which
produces
a
three
dimensional
appearing
image
of
a
living
cell
or
tssue.
Fluprescence
microscope
Used
to
study
localization
of
fluorescent
molecules
(such
as
antibodies
specific
to
certain
molecules)
adhering
to
cellular
swuctures.
Electron
Microscopes
Use
a
beam
of
electrons
(instead
of
light)
that
are
magnified
and
focused
on
a
photographic
plate
by
means
of
electromagnets.
’
Scanning
electron
microscope
Analogous
to
the
dissecting
light
microscope.
It
gives
an
image
of
the
surface
of an
object.
Transmission
electron
microscope
Analogous
to
the
compound
light
microscope.
The
object
is
thinly
sliced
and
reated
with
heavy
metal
salts
w
impy&:we
COnLrast,
Resolving
Power
The
ability
to
distinguish
two
objects.
Identification
of
parts
of
a
campout::d
light
microscope
Evepiece:
Topmost
series
of
lenses
through
which
an
object
is
viewed,
Body
wbe:
Holds
nosepiece
at
one
end
and
eyepiece
ot
the
other
end:
conducts
light
rays
Arm:_
Supporis
upper
parts
and
provides
carrying
handle
Noge
pisce:
Revulv‘ing
device
that
holds
objectives
Scanning
power
gbiective:
holds
4x
lens
used
to
view
the
wh(;la
slide
Low
power
objective:
Holds
10x
lens
used
10
view
the
subject
in
greater
detail
High
power
objective:
Holds
20x
lens
used
10
view
the
object
in
even
greater
detail
Stage:
Holds
and
suppors
microscape
slides
Coarse
adiustment
knob:
Knob
used
10
bring
object
into
approximate
focus:
used
only
with
low
power
objective
Fine
adjusument
knob;
Knob
used
to
bring
object
into
final
adjustment
Diaphram:
Conwrols
amount
of
illumination
used
W
view
the
object
Condenser:
Focuses
the
light
{concentrates
light)
Light
source:
A
mirror
that
reflects
light
from
a
lamp
up
through
the
object
4
/9
Anatomy
of
a
Microscope
Hlumination
Brightness
Miustmem\
3
s
Focusing
the
microscope
-
Low
Power
1.
With
the
coarse
adjustment
knob,
raise
the
nosepiece
unril
it
stops.
2.
Place
a
slide
of
the
lerter
e
on
the
stage
and
stabilize
it
with
the
clips.
Center
th::
&
as
best
you
can
on
the
stage.
3.
Make
sure
the
10x
objective
is
in
place;
then
as
you
look
from
the
slide
decrease
the
distance
between
the
stage
and
the
nosepiece,
until
the
nosepiece
is
no
closer
than
1/8-inch
above
the
slide.
4,
Looking
into
the
ocular
(eyepiece),
rotate
the
diaphram
@
gwe
the
maximum
amount
of
11ght
5.
Slowly
increase
the
distance
between
the
stage
and
the
noscpxacs
using
the
coarse
adjustment
knob
undl
the
object
-
in
this
case,
the
letter
@
-
comes
into
view
or
focus.
6.
Once
the
object
is
in
focus,
it
may
be
necessary
to
cdjust
the
amount
of
light.
To
create
shadows,
rotare
the
diaphram
slighdy.
-
Inversion
]
1.
In
the
space
provided
here
draw
the
letter
e
as
it
appears
on
the
slide
(look
from
the
side,
not
through
the
eyepiece,
2.
Next
to
this
draw
the
letter
e
as
it
appears
when
you
look
through
the
eyepiece.
What
differences
do
you
norice
?
Inversion
refers
to
the
fact
that
the
image
is
not
only
inverted
but
it
is
also
reversed.
Move
the
slide
to
the
right.
Which
way
does
the
image
appear
to
move
?
Focusing
the
microscope
-
High
Power
Compound
light
microscopes
are
parfocal;
that
is,
once
the
object
is
in
focus
with
low
power,
it
should
also
be
in
focus
with
high
power.
1.
Make
sure
the
lerter
¢
is
centered
on
the
stage
beneath
the
low
power
objective.
2,
Move
the
high
power
objeanv&
into
place
by
turning
the
nosepiece.
3.
If
any
adjustment
is
needed
use
only
the
FINE
adjustment
knob
with high
power.
Qn
your
draw-
ing
of
the
letter
e
as
it
was
observed
with
low
power,
draw
a
circle
around
the
pordon
of
the
letter
that
you
are
now
seeing
with
high
power
magnification.
When
you
have
finished
your
observations
of
this
slide
(or
any
slide),
rotate
the
nosepiece
until
the
low
power
objective
clicks
in
place
and
then
remove
the
slide
from
the
stage.
4,
The
following
rules
should
also
be
observed:
a.
Have
both
eyes
open
when
looking
through
the
flyepxate.
b,
The
low
power,
or
scanning,
objective
should
be
in
position
both
at
the
beginning
and
the
end
of
use,
¢.
Use
only
lens
paper
for
cleaning
the
lens,
d.
Do
not
tlt
the
microscope.
e.
Keep
the
siage
clean
and
dry
to
prevent
rust
and
corrosion,
f.
Do
not
remove
pans
of
the
microscope.
g.
Keep
the
microscope
dust
free
by
covering
it.
h.
Report
any
malfunctioning.
i,
Carry
the
microscope
with
one
hand
on
the
arm
and
one
hand
under
the
base.
A/
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Total
Magnification
Total
magnification
is
calculated
by
multiplying
the
magnification
of
the
ocular
lens
by
the
magnifi-
|
cadon
of
the
objective
lens.
What
is
the
total
magnificadon
of
Qcular
lens
Objecdve
lens
Total
Low
power:
X
=
High
power:
X
=
Depth
of
Focus.
Qbtain
a
slide
of
three
or
four
colored
threads
mounted-together.
With
low
power,
find
a
point
where
the
threads
or
hairs
cross.
Slowly
focus
up
and
down.
Notice
that
when
one
thread
is
in
focus,
the
others
seem
blurred.
Determine
the
order
of
the
threads
and
complete
the
following
table.
Depth
Thread
color
Top
Middle
Botom
The
vertical
digtance
that
remains
in
focus
at
one
tme
is
called
the
depth
of
Focus.
Switch
to
high
power
and
notice
that
the
depth
of
focus
is
more
shallow
with
high
power
than
with
low
power.
Observations
Wet
Mount
Often
it
is
necessary
to
prepare
a
specimen
for
observation.
In
such
cases,
the
object
should
always
be
viewed
as
a
wet
mount.
A
wet
mount
i$
prepared
by
placing
a
drop
of
liquid
on
a
slide,
or,
if
the
material
is
dry,
by
placing
it
directly
on
the
slide
and
adding
a
drop
of
water
or
stain.
The
mount
is
then
covered
with
a
cover
slip,
as
illustrated
in
the
figure
below.
A,
Ada
a
drop
of
water
B,
Place
the
specimen
C.
Place
the
eage
of
a
coverslip
.
Slowly
lower
’r’;!n"";‘:;"“’
'
o
a
slide
in
tha
water,
G
the
glige
50
that
it
1ouches
o
Qre’véflii
orraing
@
@
shee.
the
adge
of
iNe
water.
trapping
air
bubbles.
7
1/1)
Human
Epidermal
Cells
GENTLY
scrape
the
inside
of
your
cheek
with
a
clean,
flac
toothpick
and
place
the
scrapingson
a
.
clean
dry
slide.
Add
a
drop
of
methylene
blue
and
cover
with
a
caver
slip.
Observe
under
the
micro-
scope.
Locate
the
nucleus,
a
central
round
body
in
each
cell.
In
the
space
provided
below
draw
the
image
that
you
see
in
the
microscope:
Onion
Epidermal
Cells
With
a
scalpel
strip
a
small
thin
transparent
layer
of
cells
from
the
inside
of
an
onion
leaf,
Place
it
gently
on
a
clean
glass
slide
and add
a
drop
of
methylene
blue.
Cover
with
a
cover
slip
and
observe,
Locate
the
cell
wall
and
the
nucleus
near
the
cell
wall.
In
the
space
provided
below
draw
the
image
that
you
see
in
the
microscope:
Note
some
differences
between
the
plant
cell
and
the
animal
cell
Differences
Plant
Cell
Animal
cell
Shape
Cell
Wall
Other
AL
The
Stereoscopic
Dissecting
Microscope
The
stereoscopic
microscope,
usually
called
a
dissecting
microscope.
differs
from
the
compound
microscope
in
that
it
has
two
(rather
than
one)
objective
lenses
for
each
magnification.
This
type
of
microscope
always
has
two
oculars.
Essentially,
the
stereomicroscope
is
two
microscopes
in
one,
the
great
advantage
of
this
instrument
is
that
objects
can
be
observed
in
three
dimensions.
Because
the
alignment
of
the
two
microscopes
is
critical.
the
resolution
and
magnification
capabilitics
of
a
stercoscopic
microscope
are
less
than
in
a
compound
microscope.
Stereoscopic
microscopes
are
most
often
used
for
the
microscopic
dissection
of
specimens.
The
light
source
may
come
{rom
above
the
specimen
and
be
reflected
back
into
the
microscope;
the
stage
may
be
an
opaque
plate,
white
on
one
side
and
black
on
the
other.
Magnifications
on
this
type
of
microscope
usually
range
from
4X
to
50X.
The
oculars
can
be
adjusted
for
individual
eye
spacing
and
for
focus,
as
in
the
compound
binocular
microscope.
There
is
only
one
focus
control,
a
coarse
adjustment
knob.
Set
up
your
dissecting
microscope
with
reflected
light.
Place
your
hand
on
the
stage
and
observe
the
nail
on
your
index
finger,
Move
your
hand
so
the
image
travels
to
the
right
and
down.
How
does
this
image
movement
correspond
to
actual
movement?
Your
instructor
may
have
a
supply
of
flowers,
seeds,
or
dead
insects
to
examine
with
the
stereoscopic
microscope.
Experiment
with
it
and
learn
to
adjust
lighting
and
magnification
for
best
results.
Questions
to
be
answered
1.
Which
objective
should
always
be
in
place,
both
when
beginning
to
use
the
microscope
and
when
putting
it
away?
2.
Which
objective
is
in
focus
at
the
greatest
and
safest
distance
from
the
slide?
3.
A
total
magnification
of
100X
requires
the
use
of
the
10X
ocular
lens
with
which
objective?
4.
If
the
letter
€
is
maved
to
the
right,
in
what
direction
does
it
appear
to
move
in
the
field
of
view?
5.
A
microscope
is
called
compound
when
it
has
more
than
one
set
of
what?
6.
What
part(s)
of
a
microscope
regulates
the
amount
of
light?
~
What
word
is
used
to
indicate
that
if
the
object
is
in
focus
at
low
power,
it
will
also
be
in
focus
with
high
power?
8.
Describe
the
differences
between
a
compound
light
microscope
and
a
dissecting
(
stereascopic)
microscope.
In
what
situations
would
you
use
a
stereoscopic
microscope?
9.
How
do
animal
cells
differ
from
plant
cells?
10.
Describe
the
relationship
between
magnification,
diameter
of
the
field
of
view.
and
the
bri
ghtness
of
the
field
of
view
for
the
scanning,
low-
and
high-power
objectives
on
the
compound
microscope.
1/4
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Lab
#2
BIOLOGY
115
Laboratory
2
MITOSIS
Mitotic
cell
division
Mitotc
cell
division
is
necessary
o
the
growth
and
repair
of
multcellular
organisms.
It
is
also
a
form
of
asexual
reproducron
for
cukaryotc
single-celled
organisms.
Before
dividing,
a
cell
doubles
all
its
components,
inéluding
the
organelies
such
as
mitochondria,
ribosomes,
and
centrioles
if
present.
Because
of
DNA
replication,
each
chromosome
in
the
nucleus
is
made
up
of
a
pair
of
chromatids
held
together
at
the
centromere.
The
illustration
below
represents
a
chromosome
as
it
would
appear
just
before
nuclear
division.
Please
label
the
chromosome
drawn
below:
Mitosis
is
a
nuclear
division
thar
results
in
two
daughter
nuclei,
each
with
the
same
number
and
kinds
of
chromosomes
as
in
the
nucleus
of
the
mother
cell
(the
mother
cell
is
the
¢zil
that
divides;
the
daughter
cells
are
the
resulung
cells).
Mitosis
Model
NUCLEAR
MEMBRANE
NUCLEOLLS
RUCLEQLUS
NUCLEAR
MEMERANE
CHAOMATIN
CHNOMATIN
Interphase
During
this
stage,
replication
of
chromatin
material
(DNA)
occurs.
The
Nucleolus
is
present.
The
Nuclear
membrane
is
clearly
visible.
11
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HUCLEAR
MEMBMANE
-
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CHAOMOSOME
CHACNOIOME
ANIMAL
Prophase
The
nuclear
envelope
is
in
the
process
of
disappearing
s
a
spindle
apparatus
appears.
DNA
¢oils
tghtly
to
form
visible
chromosomes
which
appear
rod
shaped.
The
nucleolus
disappears.
In
animal
cells,
a
pair
of
centrioles
(not
visible
on
these
slides)
scpa.taw
and
with
their
ncwly
formed
asters
(visible)
move
10
cach
pole.
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:
i
*
-
A
LR
@
*,
*
*
*
e
)
‘
LR
,
!
?
[
*s
r
1
*ee
e
CHAQMOSOME
=
-
{
H
-
-
-
e
&
Metaphase
.
A
fully
formed
spindle
is
visible
which
stetches
from
pole
to
pole.
Each
chromosome
is
attached
to
a
spindle
fiber
at
the
centromere
(Kinetochore),
and
all
the
chromosormes
are
lined
up
at
the
equator.
12
A/
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et
a
s
e
e
LA
R
T
S
T
)
i
Anaphase
The
centromeres
divide;
the
chromatids
separate
(now
consisting
of
one
DNA
molecule
cach)
and
a
set
moves
toward
ecach
pole.
-
w
.
x
¢
M
R
EHEEw
-
B
l‘;x,*;
ANIMAL
Telophase
The
chromosomes
have
clustered
together
near
the
spindie
poles,
where
new
nuclear
envelopes
develop
around
them,
‘
The
spindle
slowly
dissolves.
In
aniral
cells,
cytokinesis
occurs
with
a
cleavage
furrow
dividing
one
cell
into
two
cells.
In
plant
cells,
cytokinesis
occurs
with
the
farmarion
of
a
cell
plate.
"3
9
/m
L
el
»
s
)
.h,
1
lt‘l&t{'!l‘:’:
y
T
';&;‘
[
S
A
B
.
¥o.o%
*
®
9
4
L
R
-
*
x
tt:lh.
*
ANIMAL
}
’
Interphase
(Daughter
cells)
i
This
is
the
same
as
interphase,
but
these
cells
are
smaller
and
less
mature
than
the
original
cell
Allium
(Onion)
Root
Tip
Slide
|
o
In
plants,
the
root
tip
contains
tissue
that
is
forever
dividing
and
producing
new
cells.
Examine
the
prepared
slide
of
onion
root
dp
cells
undergoing
mitotic
cell
division,
Find
the
various
stages
of
mitosis
as
illustrated
below:
Interphase
T—Telophase
.
¥~
=
Anaphase
Probhase
A
s
s
RN
O
~
i
4kl
terph
3
nierpnhase
Prophase
Metaphase
Anaphase
|
G
Ry
L
Telophase
kg
s
L
g
e
|
1
A
Duration
of
Mitotic
Stages
One
basic
assumption
of
this
exercise
is
that
the
more
cells
there
are
in
a
particular
stage,
the
longer
the
duration
of
that
stage,
A
second
assumption
is
that
the
duration
from
start
to
finish
(all
5
stages)
is
24
hours.
1.
Select
a
region
of
the
root
tip
that
scems
to
have
dividing
cells
scatered
regularly
throughout
it,
Once
the
ficld
has
been
chosen
the
slide
should
not
be
moved,
and
only
the
cells
within
that
ficld
should
be
examined
2.
In
this
field,
count
the
numnber
of
cells
in
your
field
and
the
number
of
nuclei
in
prophase,
metaphase,
anaphase
and
telophase.
Record
these
numbers
in
the
tble
on
your
Report
Sheet.
t
3.
Count
the
number
of
cells
undergoing
mitotie
cell
division
and
subtract
this
number
from
the
total
number
of
cells.
This
gives
you
the
number
of
nuclei
in
interphase.
Record
this
number
in
the
table.
Total
mitotic
figures
=
Total
number
of
cells
-
ol
mitotic
figures
=
|
nuclel
in
interphase,
The
duration
of
¢ach
mitoric
stage
may
now
be
estimated
using
the
following
equation:
duraton
of
miwde
stage
=
pumber
of
cells
in
a
stage
x
24hours
x
&Ominutes
1otal
number
of
cells
lhour
Whitefish
Blastula
The
blasmla
is
an
early
embryonic
stage
in
the
development
of
an
animal
from
the
fertilized
egg.
Examining
a
prepared
slide
will
emphasize
the
differences
between
plant
and
animal
cell
mitosis.
You
may
note
that
in
animal
cells,
the
centrioles
divide
and
the
spindle
arises
between
the
two
pairs
of
centrioles.
By
metaphase,
cach
pair
of
centrioles
has
migrated
1o
an
opposite
end
of
the
cell,
and
the
spindle
fibers
range
between
them.
Each
pair
of
centrioles
is
ar
the
center
of
radially
arranged
short
fibers
called
an
aster.
-
3
Telophase,
.
@
cytokinesis
’
‘0:._
.'
R
i
15
KI5
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Mitosis
Lab
Name:
Mitosis-Draw
what
you
see
for
each
stage
Plants
(onion
root
tips)
Animals
(whitefish
blastula)
I
U
——
g
2
e
i
-
l;
8
T
2
(-
c
0O
—
=
Q
wn
O
N
o
o
o
Q
Vs
©
N
o
(O
=
(b
=
Q
)]
o
i
o
M
C
<
Q
wn
©
i
o
O
Q
|_
Name
Number
of
cells
in
field
Number
of
nuclei
in;
Total
mitotic
figures
Nuclei
in
interphase
1.
In
your
own
words,
describe
what
happens
in
the
following
stages
of
mitosis:
a.
Prophase
Prophase
Metaphase
Anaphase
"Telophase
Mitosis
Lab
Bio
115
b.
Metaphase
¢.
Anaphase
17
t
Y.
Telophase
3.
Why
are
whitefish
blastula
and
onion
root
tips
used
to
study
mitosis?
4.
What
is
the
duration
of
each
mitotic
stage
estimated
in
the
onion
root
tip?
a.
Prophase
:
b.
Metaphase
¢.
Anaphase
d.
Telophase
5.
Describe
what
happens
during
interphase.
6.
How
is
mitosis
different
in
plants
and
animals?
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Lab
#3
Biology
115
Human
Genetic
Traits
Laboratory
3-
N
*As
a
reminder,
phenotypes
are
a
description
of
the
ame
)
=
trait
(ex.
widow's
peak)
and
genotypes
are
a
combination
of
two
alleles
(ex.
RR,
Rr,
or
rr)
Define:
phenotype__
genotype,
1.
Tongue
'rolling:
The
tongue
rolling
allele
(R)
is
dominant
to
the
non-rolling
allele
(r)
Your
phenotype
.
Partner's
phenotype
Your
possible
genotype
|
Partner's
possible
genotype
If
your
mother
was
a
non
tongue-rolier
then
your
genotype
would
have
to
be
,
and
if
your
partner's
mother
was
a
non
tongue-roller
then
his/her
genotype
would
have
to
be
___
2.
Widow's
Peak:
The
widow's
peak
allele
(W)
is
dominant
to
the
straight
hairline
allele
(w)
Your
phenotype
.
)
Partner's
phenotype
Your
possible
genotype
|
Partner's
possible
genotype
3.
Free
earlobe:
The
free
earlobe
allele
(E)
is
dominant
to
the
attached
earlobe
allele
(e)
Your
phenotype
)
Your
possibie
genotype
Does
your
father
have
free
earlobes?
Does
your
mother
have
free
earlobes?
‘
Do
your
brothers
and
sisters
have
free
earlobes?
What
is
your
complete
genotype
for
earlobes?
How
do
you
know?
4.
Taste
PTC:
The
PTC
tasting
allele
(T)
is
dominant
to
the
inability
to
taste
PTC
allele
(t)
Can
you
taste
this
chemical?
|
Can
your
partner
taste
PT1CY
Your
possible
genotype
Partner's
possible
genotype
|
Based
on
you
and
your
partner's
genotypes,
list
all
possible
genotypes
that
your
child
could
have
for
the
ability
to
taste
PTC.
20
4/4
5
Mitch-hiker's
thumb:
The
straight
thumb
allele
(Hi)
is
dominant
to
the
hitchhiker's
thumb
allele
(hi)
Your
phenotype
Partner's
phenotype
Your
possible
genotype
Partner's
possible
genotype
6.
Bent
little
finger:
The
bent
little
finger
allele
(Bf)
is
dominant
to
the
straight
little
finger
allele
(bf)
Your
phenotype
)
Parther's
phenotype
Your
possible
genotype
Partner's
possible
genotype
7.
Mid-digital
hair:
The
hair
on
the
mid-digit
allele
(M)
is
dominant
to
the
no
hair
allele
(m)
Your
phenotype
Partner's
phenotype
Your
possible
genotype
Partner's
possible
genotype
_
8.
Facial
dimples:
The
dimples
allele
(D)
is
dominant
to
the
no
dimples
allele
(d)
Your
phenotype
Partner's
phenotype
Your
possible
genotype
Partner's
possible
genotype
.
9.
Hallux
length:
The
short
big
toe
allele
(Ha)
is
dominant
to
the
long
big
toe
allele
(ha)
Your
phenotype
_
Partner's
phenotype
Your
possible
genotype
Partner's
possible
genotype
The
short
index
finger
allele
(S®)
is
dominant
to
the
long
index
finger
allele
(Sb)
in
10.
Index
finger
length:
males,
but
in
females
the
long
index
finger
is
dominant
Your
sex
|
Partner's
sex
.
Your
phenotype
,,
Partner's
phenotype
Your
possible
genotype
__________
Partner's
possible
genotype
-
If
you
have
a
dominant
phehotype
for
any
of
the
above
traits,
how
could
you
determine
your
genotype?
Individuality
exercise
With
the
exception
of
identical
twins,
each
of
us
has
a
unique
genetic
endowmept.
'_Thls
can
be
illustrated
in
the
following
manner..
When
the
class
has
finished
recording
their
traits,
the_y
should
all
stand.
One
student
is
appointed
as
reader.
The
reader
reads
his
or
her
first
traft
from
the
table.
All
students
who
do
not
have
this
trait
sit
down.
The
reader
the_r}
reaqs
|
hgs
or
her
second
trait
and
again
all
others
who
do
not
have
this
trait
sit
down.
Continue
in
this
manner
until
only
the
reader
remains
standing.
This
procedure
can
be
repeated
several
times
using
a
different
reader
each
time.
;
21
24
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1.
Maple
syrup
urine
disease
is
a
human
recessive
disorder.
a.
|f
Kim
has
this
disorder,
what
is
her
genotype?
b.
How
do
you
know
this?
2,
Recall
that
widow's
peak
and
free
earlobes
are
dominant
traits.
Latoya
has
a
widow's
peak
and
free
earlobes.
Dennis
has
a
widow's
peak
and
attached
earlobes.
They
have
two
children:
one
has
a
widow's
peak
and
attached
earlobes,
and
the
other
has
a
straight
hairline
and
attached
earlobes.
a.
What
are
the
genotypes
of
Latoya
and
Dennis?
b.
Considering
the
earlobes
only,
what
fraction
of
their
children
would
be
expected
to
have
free
earlobes?
c.
Does
the
fact
that
they
have
no
children
with
free
earlobes
mean
that
earlobe
inheritance
is
not
a
simple
Mendelian
trait?
Explain
.
22
.Z-/
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Widow's
peak
Tongue
roller
Free
ear
lobe
Attached
ear
lobe
Hitchhiker's
thumb
]
%
Hitch-hiker's
thumb
Bent
little
finger
HUMAN
PHENOTYPES
1.
Carolina
Biological
Supply
Company,
Burlington,
North
Carolina
27215
ot
St
Printed
n
LLSA.
amg%wmmumwmx
/
),"
Rearoduction
of
all
or
anv
nart
of
this
sheat
without
writien
pereission
Irom
the
copyright
holdes
(s
unlawful,
‘
/‘[
x.e'l‘
.
f
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.v
A
l
'
\
l'.
W\
Mid
digital
Hair
Mid
digital
Hair
Absent
Present
2
id
>
K
-
J
|
s
Mid-digital
hair
Dimples
Long
Hallux
e
M
___________________________________________
v
A
)
_
Short
hallux
Short
index
finger
HUMAN
PHENOTYPES
2.
@fi
Carolina
Biological
Supply
Company,
Burlington,
North
Carolina
27215
Biophoto*
Sheet
Printed
in
(LA,
&
1985
Carola
Blological
Sugply
Compary
4832
.
/-
s
Fleproduction
of
all
or
any
pard
of
this
sheet
without
witlien
potmigsion
fram
the
copyright
holder
is
unlawlul.
N
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Lab
#4
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WARDS
Si‘mulated
ABO
and
Rh
Blood
Typing
Student
Study
and
Analysis
Sheets
36
W
0019
Note:
In
response
to
the
concerns
of
many
science
educators,
WARD'’S
has
developed
an
alternative
biood
typing
activity
that
does
not use
real
blood,
Students
will
follow
the
same
procedure
used
to
type
actual
human
blood
and
obitain
results
that
closely
approximate
thase
obtained
by
real
blood
typing.
Iintroduction
Around
1900,
Karl
Landsteiner
discovered
that
there
are
at
least
four
different kinds
of
hurnan
blood,
deter-
mined
by
the
presence
or
absence
of
specific
agglutinogens
(agglutinating
antigens)
on
the
surface
of
red
blood
cells
(erythrocytes),
These
antigens
have
been
designated
as
A
and
B.
Antibodies
against
antigens
A
or
B
begin
to
build
up
in
the
blood
plasma
shortly
after
birth,
the
levels
peak
at
about
eight
to
10
years
of
age,
and
the
antibodies
remain,
in
declining
amounts,
throughout
the
rest
a
person’s
of
life.
The
stimulus
for
anti-
body
production
is
not
clear;
however,
it
had
been
proposed
that
antibody
production
is
initiated
by
minute
amounts
of
A
and
B
antigens
that
may
enter
the
body
through
food,
bacteria,
or
other
means.
Humans
nor-
mally
produce
antibodies
against
those
antigens
that
are
not
on
their
erythrocytes:
A
person
with
A
antigens
has
anti-B
antibodies;
a
person
with
B
antigens
has
anti-A
antibodies;
a
person
with
neither
A
or
B
antigens
has
both
anti-A
and
anti-B
antibodies;
and
a
person
with
both
A
and
B
antigens
has
neither
anti-A
nor
anti-B
antibodies.
Blood
type
is
based
on
the
antigens,
not
the
antibodies,
a
person
possesses.
The
four
blood
groups
are
types
A,
B,
AB,
and
O,
Blood
type
O,
characterized
by
the
absence
of
A
or
B
agglu-
tinogens,
is
the
mast
common
in
the
United
States,
in
45%
of
the
population.
Type
A
is
next
in
frequency,
found
in
39%
of
the
population,
The
incidences
of
types
B
and
AB
are
12%’and
4%
respectively.
ABO
System
Antigens
on
Antibodies
:
Blcod
Erythrocytes
in
Plasma
.
Can
Give
|
Can
Recelve
Type
|
(Agglutinogens)
{Agglutinins)
Blood
To
Blood
From
A
,
A
Anti-B
A,
AB
Q,A
B
B
Anti-A
,
B,
AB
Q,B
AB
Aand
B
°
|
Nelther
Anti-A
nor
Anti-B
AB
O,
A,
B,
AB
0
Neither
A
nor
B
Both
Anti-A
and
Anti-B
O,
A
B,
AB
O
Copymastes.
PUmsion
granted
to
make
unkmited
copies
fr
e
in
any
one
©
1998
WARD'S
Natwral
Science
Estahlishment,
Inc.
schoot
bulding.
For
educational
use
orily,
Not
for
commertial
use
o
resaie.f
All
Rights
Rescrved,
D/
A
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4
Process
of
Agglutination
There
is
a
simple
test
to
determine
blood
type,
performed
with
antisera
containing
high
levels
of
anti-A
and
anti-B
agglutinins.
Severat
drops
of
each
kind
of
antiserum
are
added
to
separate
samples
of
blood.
If
agglu-
tination
(clumping)
occurs
only
in
the
suspension
to
which
the
anti-A
serum
was
added,
the
blood
type
is
A,
If
agglutination
occurs
only
in
the
anti-8
mixture,
the
blood
type
is
B.
Agglutination
in
both
samples
indicates
that
the
blood
type
is
AB.
The
absence
of
agglutination
in
any
sample
indicates
that
the
blood
type
is
O,
Agglutination
Reaction
of
ABO
Blood-Typing
Sera
Reaction
Blood
Type
Anti-A
Serum
Anti-B
Serum
|
Agglutination
No
Agglutination
Type
A
No
Agglutination
Agglutination
Type
b
__Agglutination
@ufinatim
Type
AB
No
Agglutination
|
No
Agglutination
Type
O
Importance
of
Blood
T).;ping
As
noted
in
the
tabie
above,
people
can
receive
transfusions
of
only
certain
blood
types,
depending
on
the
type
of
blood
they
have.
if
incompatible
blood
types
are
mixed,
erythrocyte
destruction,
agglutination
and
other
problems
can
occur.
For
instance,
If
2
person
with
Type
B
blood
is
transfused
with
bicod
type
A,
the
recipient’s
anti-A
antibodies
will
attack
the
incoming
type
A
erythrocytes.
The
type
A
erythrocytes
witl
be
agglutinated,
and
hemoglobin
will
be
released
into
the
plasma.
In
addition,
incoming
anti-B
antibodies
of
the
type
A
blood
may
also
attack
the
type
B
erythrocytes
of
the
recipient,
with
similar
results.
This
problem
may
not
be
serious,
unless
a
large
amount
of
blooad
is
transfused.
.
The
ABQ
blood
graups
and
other
inherited
antigen
characteristics
of
red
blood
cells
are
often
used
in
medico-
legal
situations
involving
identification
of
disputed
paternity.
A
comparison
of
the
blood
groups
of
mother,
child,
and
alleged
father
may
exclude
the
man
as
a
possible
parent.
Blood
typing
does
not
prove
that
an
indi-
vidual
is
the
father
of
a
child;
it
merely
indicates
whether
or
not
he
is
a
possible
parent,
For
example,
a
child
with
a
blood
type
of
AB,
whose
mother
is
type
A,
could
not
have
as
a
father
a
man
whose
blood
type
is
O.
The
Genetics
of
Blood
Types
'
The
hurman
blood
(A,
B,
AB,
and
Q)
are
inherited
by
multiple
alleles—three
or
more
genes
that
occupy
a
single
locus
on
a
chromosome.
Gene
1
codes
for
the
synthesis
of
antigen
(agglutinogen)
A,
gene
[B
codes
for
the
production
of
antigen
B
on
the
red
blood
celis,
and
gene
i
(i®)
does
not
produce
any
antigens.
The
phenotypes
listed
in
the
tabie
below
are
produced
by
the
combinations
of
the
three
different
alleles:
14,
18,
{0,
When
genes
B
and
|4
are
present
in
an
individual,
both
are
fully
expressed.
Both
IA
and
(¥
are
dominant
over
i9;
the
genotype
of
an
individual
with
blood
type
O
must
be
{50,
:
Phenotypes
|
Possible
Genotypes
.
A
AA
A
{or
19)
B
|B{8
I8
(19)
Q
LK
Use
1A
fnr'ani:igen
A,
I8
for
anfig;’en‘
B,
i
or
19
for
no
antigens
present
Genes
I*
and
8
are
dominant
over
i
(I9)
AB
blood
type
results
when
both
genes
{A
and
I®
are
present
Copymyster,
&mmflmwmmmWMmfiafmmmwme
;
©
1998
WARD'S
Natural
Science
Establishment.
Inc.,
&hool
building.
Fod
ediycanonal
use
only.
Not
for
comenercial
use
or
resale.
All
Rights
Reserved,
-
/
.
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3
Rh
System
In
the
period
between
1900
and
1940,
a
great
deal
of
research
was
done
to
discover
the
presence
of
other
antigens
in
hurman
red
blood
cells.
In
1940,
Landsteiner
and
Wiener
reported
that
rabbit
sera
containing
anti-
bodies
for
the
red
blood
cells
of
the
Rhesus
monkey
would
agglutinate
the
red
blood
cells
of
5%
of
white
humans.
These
antigens,
six
in
all,
were
designated
as
the
Rh
(Rhesus)
factor:
they
were
given
the
letters
C,
¢,
D,
d,
£,
and
e
by
Fischer
and
Race.
Of
these
six
antigens,
the
D
factor
is
found
in
85%
of
Caucasians,
949%
of
African
Americans,
and
99%
of
Asians.
An
individual
who
possesses
these
antigens
is
designated
Rh+;
an
indi-
vidual
who
lacks
them
is
designated
Rh-,
The
genetics
of
the
Rh
blood
group
system
is
complicated
by
the
fact
that
more
than
one
antigen
can
be
iden-
tified
by
the
presence
of
a
given
Rh
gene.
initially,
the
Rh
phenotype
was
thought
to
be
determined
by
a
sin-
gle
pair
of
alleles.
However,
there
are
at
least
eight
alleles
for
the
Rh
factor.
To
simplify
matters,
consider
one
allele:
Rh+
is
dominant
over
Rh-;
therefore,
a
person
with
Rh+/Rh-or
Rh+/Rh+
genotypes
has
Rh+
blood.
The
anti-Rh
antibodies
of
the
system
are
not
normally
present
in
the
plasma,
but
anti-Rh
antibodies
can
be
produced
upon
exposure
and
sensitization
to
Rh
antigans.
There
are
several
ways
sensitization
can
occur-—for
example,
if
Rh+
blood
is
transfused
into
an
Rh-
recipient,
or
when
an
Rh-
mother
carries
a
fetus
who
is
Rhv+.
In
the
latter
case,
some
of
the
fetal
Rh
antigens
may
enter
the
mother’s
circulation
and
sensitize
her
so
that
she
begins
to
produce
anti-Rh
antibodies
against
the
fetal
antigens.
In
most
cases,
sensitization
to
the
Rh
anti-
gens
takes
place
toward
the
end
of
pregnancy,
but
because
it
takes
some
time
to
build
up
the
anti-Rh
anti-
odies,
the
first
Rh+
child
carried
by
a
previously
unsensitized
mother
is
usually
unaffected.
However,
if
an
Rh-
mother,
or
a
mother
previously
sensitized
by
a
blood
transfusion
or
a
previous
Rh+
pregnancy,
carries
an
Rh+
fetus,
maternal
anti-Rh
antibodies
may
enter
the
fetus’
circulation,
causing
the
agglutination
and
hemolysis
of
fetal
erythrocytes
and
resulting
in
a
condition
known
as
erythroblastosis
fetalis
(hemolytic
disease
of
the
new-
born).
To
treat
an
infant
in
a
severe
case,
the
infant’s
Rh+
blood
is
removed
and
replaced
with
Rhi--
biood
from
an
unsensitized
donor
to
reduce
the
leve!
of
anti-Rh
antibodies.
:
Artificial
Blood
At
times
it
is
difficult
to
find
a
correct
match
for
a
blood
type
of
a
person
t:equiring
a
transfusion.
It
would
be
ideal
to
have
some
type
of
artificial
blood
or
blood
substitute
that
wouldn't
need
to
be
matched
to
a
patient’s
blood
type;
it
could
save
thousands
of
lives
each
year.
Although
the
research
for
artificial
blood
and
blood
sub-
stitutes
continues,
it
may
take
years
before
one
is
available,
In
1966,
Dr.
Leland
C,
Clark,
of
the
University
of
Cincinnatl's
College
of
Medicine,
developed
the
first
arti-
ficial
blood
prototype.
This
milky
white
solution,
which
can
carry
twice
as
much
oxygen
as
blood
does,
is
a
fluorocarbon
emulsion
called
Fluosol,
It
is
made
up
of
two
fiuorocarbons,
a
number
of
salts,
water,
and
fine
particies
that
are
'/
the
size
of
erythrocytes.
Because
these
particles
are
so
small,
they
can
pass
through
narrowed
arteries
that
the
larger
erythrocytes
can’t
get
through,
making
it
an
ideal
blood
substi-
tute
for
heart
attack
and
stroke
victims;
they
would
recover
faster
and
have
less
tissue
damage.
Fluosol
has
been
approved
for
use
In
Canada,
Holland,
and
Italy.
The
US
Food
and
Drug
Administration
is
reviewing
Fluosol
for
use
in
the
United
States.
‘
Anthony
Hunt
and
colleagues
at
the
University
of
California
at
San
Francisco
are
working
with
artificial
red
blood
cells
called
neohemocytes.
Neohemocytes,
which
are
microscopic
spheres
of
hemoglobin
surrounded
by
lipids,
are
capable
of
carrying
oxygen.
These
microspheres
are
proving
to
be
a
successful
substitute
for
ery-
'
throcytes.
As
with
Fluosol,
their
small
size
allows
them
to
pass
through
restricted
vessels
that
might
not
aliow
the
passage
of
erythrocytes.
‘
Copymastes.
PErTusIOn
granted
10
make
Lobimitsd
copies
1
Lse
in
3y
e
"
[V
.
schood
building.
For
educatonal
use
anly.
Not
for
commeral
use
or
resale.
8
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Objective
To
use
WARD'S
Simulated
Blood
to
determine
the
biood
type
and
Rh
factor
of
four
individuals.
Also
to
use
a
simplified
counting
technique
to
estimate
the
number
of
red
and
white
blood
cells
per
cubic
millimeter.
A.
ABO
and
Rh
Blood
Typing
Materials
Needed
per
Lab
Group
4
Blood
Typing
Slides
8
Toothpicks
Shared
Hateriais
4
Unknown
Blood
Samples
Mr.
Smith
Mr.
Jones
Mr.
Green
Ms.
Brown
:
Simulated
Anti-A
Typing
Serum
Simulated
Anti-B
Typing
Serum
‘
Simulated
Anti-Rh
Typing
Serurmn
Procedure
1,
Label
each
blood
typing
slide:
Slide
#1:
Mr.
Smith
Slide
#2:
Mr,
jones
Slide
#3:
Mr.
Green
Slide
#4:
Ms,
Brown
2.
Place
three
to
four
drops
of
Mr.
Smith’s
blood
in
each
of
the
A,
B,
and
Rh,
wells
of
Slide
#1.
3,
Place
three
to
four
drops
of
Mr.
Jones’s
blood
in
each
of
the
A,
B,
and
Rh,
wells
of
Slide
#2.
4,
Place
three
to
four
drops
of
Mr.
Green'’s
blood
in
each
of
the
A,
B,
and
Rh,
wells
of
Slide
#3.
5.
Place
three
to
four
drops
of
Ms.
Brown's
blood
in
each
of
the
A,
B,
and
Rh,
wells
of
Siide
#4,
6.
Add
three
to
four
drops
of
the
simulated
anti-A
serum
in
each
A
well
on
the
four
slides.
7.
Add
three
to
four
drops
of
the
simulated
anti-B
serum
in
each
B
well
on
the
four
slides.
8.
Add
three
to
four
drops
of
the
simulated
anti-Rh
serum
in
‘each
Rh,
well
on
the
four
slides.
9.
Stir
each
mixture
with
a
different
clean
toothpick.
Use
only
one
toothpick
per
well
to
avoid
cross
contamination,
10,
Observe
and
record
the
results
in
Table
1.'
Analysis
Table
1
Agglutination
Reactions
Anti-A
Serum
Antl-B
Serum
__Anti-Rh
Serum
|
Blood
Type
Stide
#1:
Mr.
Smith
Slide
#2:
Mr.
jones
slide
#3:
Mr.
Green
Slide
#4:
Ms.
Brown
7Y
Copymaster,
PETVAON
Granted
10
Make
Unsmited
COpEs
for
LSe
I
3
IR
SR
TN
T
SRR
g
W
R
LY
P
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Questions
ABO
Blood
Group
1.
What
is
Mr.
Smith’s
blood
type?
What
ABQ
agglutinogens
are
present
in
his
red
blood
cells?
2.
What
is
Mr.
Green's
blood
type?
What
ABO
agglutinins
are
present
in
the
plasma
of
his
blood?
3.
What
is
Mr.
Jones’s
blood
type?
If
Mr.
jones
needed
a
transfusion,
what
blood
type(s)
could
he
safely
receive?
4.
What
is
Ms.
Brown's
binod
type?
What
blood
type(s)
could
safely
receive
her
donated
blood?
5.
Why
s
it
necessary
to
match
the
donor's
and
recipient’s
blood
before
a
transfusion?
6.
What
happens
to
red
blood
cells
that
are
agglutinated?
Copymaster.
Fermesion
granted
o
make
urinied
copes
for
use
n
any
one
s6ho0!
bullding,
For
aducational
wse
aaly,
Not
for
Commerial
wse
or
res3g.
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7.
What
is
the
difference
between
agglutinogens
and
agglutinins?
8.
How
are
ABO
blood
types
determined?
9.
Could
2
man
with
an
AB
blood
type
be
the
father
of
a
child
with
type
Q
blood?
16,
Could
a
man
with
an
O
blood
type
be
the
father
of
a
child
with
type
AB
blood?
11.
Could
a
type
B
child
with
a
type
A
mother
have
a
type
A
father?
12.
What
are
the
possible
genetic
combinations
of
a
child
whose
parents’
blood
types
are
A
and
B?
7/6
Capymaster,
Pemisson
grarked
10
Make
LBk
(O
106
Lse
1
any
one
Sehool
Dulding.
FOr
sCucatonst
use
only,
Not
fSe
commerciat
use
of
.
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Lab
#5
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NEO/Sc
o
NP
STUDENT
&
Name
GUIDE
TEACHER/SECTION
'
DATE
yyyyyyyy
Karyotyping
of
Chromosomes
Activity
Model
‘
Objectives
)
Prepare
various
human
karyotypes
of
chromosomes.
Predict
genetic
disorders
using
karyotypes.
E
Understand
the
role
chromosomes
play
in
the
process
of
reproduction,
Simulate
the
phases
of
Meiosis
1
and
Il
Demonstrate
how
sexual
reproduction
produces
genetic
diversity.
‘
Background
)
Diseases
that
run
in
families
are
calied
“genetic
diseases?
What
is
the
risk
of
inheriting
a
genetic
disease?
Why
do
some
diseases
appear
more
often
in
males
than
in
females?
Scientists
use
family
histories,
called
“pedi-
grees,
as
well
as
images
of
chromosomes
and
molecular
studies
of
DNA,
to
answer
these
and
other
questions.
*
Until
recently,
doctors
could
not
tell
whether
someone
was
at
risk
of
incurring
a
genetic
disease
until
symptoms
appeared.
Gene-screening
techniques
have
now
made
it
possible
to
determine
whether
a
person
is
predisposed
to
certain
diseases.
These
tests
can
also
confirm
the
presence
of
a
specific
gene
defect
or
mutation
within
an
individual
or
a
family.
(Genetic
screening
involves
examining
a
person’s
DNA
in
order
to
detect
an
abnormality
that
signals
a
disease
or
disorder,
The
defect
may
be
extrernely
small.
In
some
cases,
a
genetic
disease
is
caused
by
a
change
in,
or
deletion
of,
one
nucleotide
base.
Other
genetic
disorders
are
caused
by
large
abnormalities.“Translocations”
result
from
a
segment
of
DNA
detaching
from
one
chromo-
some
and
reattaching
to
another,"Deletions”
occur
when
Karyotyping
of
Chromosomes
Activity
Model
large
segments
of
DNA
are
missing
from
a
chromosome
altogether.
Another
type
of
large
genetic
error
is
known
as
an
"Inversion’
This
results
from
a
segment
of
DNA
becoming
detached
from
a
chromosome,
turning
1809,
and
reattaching
itself
to
the
same
chromosome.
5till
another
abnormal
condition
is
called
a
“duplication?
whereby
a
segment
of
DNA
is
copied
more
than
once,
end
to
end,
resulting
in
more
than
one
gene
of
the
same
type
on
the
same
chromosome,
The
normal
chromosome
number
in
humans
is
46,
meaning
that
you
have
a
total
of
23
homologous
pairs
of
chromosomes.
You
inherited
one
set of
23
chromo-
somes
from
your
mother,
and
a
corresponding
set
of
23
chromosomes
from
your
father.
Chromosomes
1
through
22
are
called
“autosomes’,
because
they
have
genes which
code
for
traits
other
than
the
sex
of
the
individual.
The
sex
chromosomes
(#23)
come
in
two
forms,
X
and
Y.
in
humans,
inheriting
two
X
chromaosomes
results
in
a
fernale
child,
while
inheriting
one
X
and one
Y
results
in
a
male.
Other
genes
are
located
on
the
sex
chromosomes
also,
with
the
X
chromosome
having
more
genes
than
the
Y
chromosome.
Those
traits
coded
for
by
genes
on
the sex
chromosomes
are
called
“sex-linked
traits’
Meiosis
is
the
process
by
which
eggs
and
sperm
are
produced.
In
order
to
keep
the
chromosome
number
constant
at
46
from
generation
to
generation,
each
egg
or
sperm
must
contain
only
23
chromosomes,
At
fertilization,
the
total
number
of
46
chromosomes
is
restored,
and
the
embryo
inherits
a
complete
set
of
genetic
instructions
from
its
parents.
During
meiosis,
chromosome
pairs
line
up
and
separate
into
daughter
cells,
Sometimes,
this
separation
doesn’t
occur
normally,
and
a
daughter
cell
with
either
too
many
or
too
few
chromosomes
can
result,
This
process
is
called
"nondis-
junction’
and
can
occur
during
the
production
of
either
eggs
or
sperm.
These
defective
cells
may
still
participate
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NED/Sc
NEO
GU!DEI’F’
TEACHER/SECTION
DATE
4
SCI
A
daughter
cell
which
recelves
the
extra
chromosome
copy
wili
end
up
with
a
total
of
3
copies
of
that
chromo-
some
after
fertilization.
This
s
calted
a
“trisomy?
The
most
cormmon
human
trisomy
is
Trisomy
21
which
results
in
Down
syndrome,
which
is
designated
by
scientists
as
(47,
XY,
+21).
The
incidence
of
this
disorder
is
approximately
1
in
600
live
births,
It
causes
mental
retardation,
distinct
facial
features,
and
heart
defects.
The
daughter
cell
receiving
no
coples
of
a
chromosome
during
melosis
will
end
up
with
only
a
single
copy
of
that
chromosome
following
fertilization.
This
is
called
a
“monosomy’
Most
trisomies
and
monosomies
are
lethal
to
the
embryo,
In
addition
to
occurrence
via
nondisjunc-
tion,
Trisomy
21
can
also
occur
through
translocation.
Part
of
the
number
21
chromosome
is
exchanged
with
chromosome
number
14.
The
person
with
such
a
genome
is
healthy,
however
he
or
she
may
produce
chromosomally
unbalanced
offspring.
:
Kleinfelter's
syndrome,
designated
(47
-XXY),
is
another
chromosomal
disorder
which
occurs
when
an
extra
X
chromosome
is
inherited
from
either
parent,
The
incidence
of
this
disorder
is
1
per
1000
males
with
an
increased
risk
at
increased
maternal
age,
People
with
this
disorder
are
infertile
males
with
some
female
physical
characteristics
due
to
poor
production
of
testosterone,
Another
common
chromosomal
disorder
is
Turmer’s
syndrome,
designated
(45,
X).
Individuals
with
this
disorder
have
only
one
X
chromosome
and
no
Y
chro-
mosome.
This
results
from
a
nondisjunction
of
the
sex
chromosomes
during
meiosis.
The
incidence
of
this
disorder
is
approximately
1
in
5,000
live
female
births.
Because
these
individuals
have
no
Y
chromosome,
they
are
females.
However,
they
lack
functional
reproductive
organs
and
have
a
range
of
abnormalities,
including
short
stature,
renal
and
cardiovascular
anomalies,
and
lower-than-average
1Q.
Karyotyping
of
Chromosomes
Activity
Model
Predictive
gene
tests
identify
people
at
risk
for
a
disease
before
any
symptoms
appear.
These
tests
identify
disor-
ders
that
run
in
families
because
the
defective
gene
is
passed
from
one
generation
to
the
next.
More
than
two
dozen
of
these
tests
are
currently
available
for
diseases
such
as
Tay-Sachs,
cystic
fibrosis,
and
Huntington’s.
Scientists
are
in
the
process
of
developing
similar
tests
for
Lou
Gehrig’s
disease,
some
forms
of
Alzheimer's,
extremely
high
cholesterol
levels,
and
some
cancers,
such
as
colon
and
breast.
To
develop
predictive
gene
tests,
scientists
study
DNA
samples
from
members
of
families
with
a
high
incidence
over
several
generations
of
a
particular
condition.
If
the
gene
itself
cannot
be
studied,
they
look
for
easily
identified
segments
of
DNA,
known
as
“genetic
markers’
that
are
consistently
inherited
by
family
members
with
the
disease
but
are
not
found
in
relatives
who
are
disease-free,
An
accurate
gene
test
can
tell
whether
an
individual
has
the
mutation
associated
with
a
particular
disease,
but
it
cannot
determine
whether
the
person
will
actually
develop
the
disease.
That's
because
many
factors
influence
the
gene’s
exprassion.
Predictive
gene
tests
deal
in
probabilities,
not
certainties.
In
some
cases,
people
who
know
they
are
predisposed
to
a
particular
disease
can
modify
their
diet,
behavior,
or
lifestyle
to
decrease
their
risk
of
developing
the
disease,
In
other
cases,
they
can
undergo
regular
screening
for
the
dis-
ease.
Many
cancers
can
be
successfully
treated
if
they
are
caught
early
enough.
In
1989,
scientists
from
all
over
the
world
began
collect-
ing
data
on
the
sequence
of
nucleotide
bases
in
human
chromosomes.
This
effort
is
known
as
the
Human
Genome
Project.
Their
goal
is
to
identify
the
function
and
map
the
location
of
all
human
genes.
This
knowl-
edge
may
help
pinpoint
genes
responsible
for
many
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NEO/S
STUDENT'S
NAME
Guipt
'
TEACHER/SECTION
AEREEPHEH
SR
PR
ESPEHP
I
OIIRI
RS
AN
I
ORI
PI
AR
-
DATE
ARd
AL
AR
A
A
i
St
L]
serious
human
diseases
and
disorders.
One
possible
benefit
of
the
Hurnan
Genome
Project
is
the
develop-
ment
of
gene
therapy
to
treat
certain
types
of
inherited
disorders.
Gene
therapy
involves
using
retro
viruses
to
insert
a
normal
human
gene
into
a
defective
chromosome,
In
order
to
identify
defective,
extra,
or
missing
chromo-
somes,
scientists
prepare
a
karyotype
-
a
visual
represen-
tation
of
an
individual’s
chromosomes
arranged
in
a
specific
pattern.
This
standard
arrangement
shows
homelegous
pairs
ordered
according
to
their
size,
shape,
banding
pattern,
and
centromere
position,
Karyotypes
have
become
increasingly
important
as
more
diseases
are
linked
to
chromosomal
abnormalities.
Scientists
who
prepare
and
study
karyotypes
are
called
“cytogeneticists”
First,
they
extract
chromosomes
from
white
blood
cells
and
expose
them
to
a
variety
of
chemicals
and
stains
to
make
the
chromosomes
easier
to
see,
Next,
they
photograph
the
cells
in
the
metaphase
stage
of
mitosis
(i.e.
division)
through
a
microscope.
The
characteristics
of
chromosomes
are
easiest
to
see
during
metaphase.
The
cytogeneticists
then
use
computer
equipment
to
arrange
the
chromosome
spread
into
a
karyotype.
Chromosome
#
ldentifying
Characteristics
‘\
1,2,3
Very
long;
centromere
in
the
center
4,5
Very
long;
centromere
not
in
the
center
6,7,8910,11,12
Long;
centromere
not
in
the
center
13,14,
15
|
Mediurm
In
length;
centromere
above
the
center
16,17,
18
Miedium
in
length;
centromere
at
or
close
to
the
center
)
-
19,20
'
Short;
centramer&..za;
or
close
m
tha
center
2122
Short;
centromere
not
in
the
center
Sex
Chrorr;l;;;mé
Xifi
inng,
;entromere
in
the
center
m
\\
Y
Is
short;
centromere
above
the
center
)
Karyotyping
of
Chromosomes
Activity
Model
(OPYMASTER:
Permission
granted
to
make
unfimited
copies.
Copy
use
confined
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educational
purpoa%s
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Bio 115
Human
Karyotyping
Each
species
has
a
characteristic
number
of
chromosomes;
for
example,
comn
cells
have
20
chromosomes,
mouse
cells
have
40,
and
dog
cells
have
78.
The
analysis
of
human
chromosomes
has
been
actively
pursued
since
it
was
first
demonstrated
that
the
normal
number
of
chromosomes
in
the
human
species
is
46.
A
karyotype
is
a
display
of
human
chromosomes
arranged
in
homologous
(matched)
pairs.
In
this
activity,
you
will
use
simulated
karyotypes
for
various
individuals
and
analyze
their
chromosome
patterns.
Human
karyotypes
are
usually
prepared
from
photographs
of
chromosome
spreads
which
have
been
fixed
and
stained
to
highlight
banding
patterns.
The
chromosomes
in
the
photograph
are
cut
apart
and
then
arranged
in
homologous
pairs.
Some
karyotypes
encountered
are
Normal
male
—
46
chromosomes
(22
pairs
of
autosomes,
1
X
chromosome
and
1
Y
chromosome.)
Normal
female
-
46
chromosomes
(22
pairs
of
autosomes,
2.
chromosomes.)
Down
syndrome
—
called
trisomy
21
is
usually
caused
by
the
presence
of
an
extra
chromosome
21
(47
chromosomes,
22
pairs
of
fiutnsam&s,
an
extra
copy
of
chromosome
21
and
two
sex
chromosomes).
The
patient
may
be
either
male
or
female.
Some
Down
syndrome
patients
are
severely
retarded
while
others
may
be
mildly
retarded.
'
Klinefelter
syndrome
~
is
associated
with
the
presence
of
an
extra
X
chromosome
in
a
male
(47
chromosomes,
2
X
and
1
Y
chromosome.)
Males
with
this
disorder
are
often
sexually
undeveloped.
Turner
syndrome
—
females
missing
one
copy
of
the
X
chromosome
(45
chromosomes,
22
autosomes,
1
X
chromosome).
These
females
are
usually
sterile.
Procedure:
On
the
worksheet
record
the
smear
number.
36
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Use
scissors
to
cut out
the
chromosomes
for
the
individual
shown
on
the
worksheet,
Match
the
homologous
pairs
of
chromosomes
and
place
them
on
the
worksheet
in
their
appropriate
space.
Use
the
line
provided
to
line
up
the
centromere
region
on
each
chromosome,
Use
the
shape,
size,
banding
patterns,
and
group
descriptions
to
arrange
the
karyotype.
Refer
to
your
text
for
guidance,
When
your
chromosomes
have
been
classified
and
you
are
confident
of
their
identity,
tape
them
to
your
worksheet
and
answer
the
questions
on
the
worksheet.
37
e
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Questions
to
answer:
1.
What
specific
meiotic
event
would
have
to
occur
to
cause
Down’s
syndrome?
2.
a.
What
sex
chromosomes
does
an
abnormal
sperm
have
to
carry
in
order
to
produce
an
XXY
offspring
when
it
combines
with
a
normal
egg?
Meiotic
non-disjunction
of
the
sex
chromosomes
in
either
parent
can
produce
a
child
with
Klinefelter's
syndrome
(XXY)
or
Turner’'s
syndrome
(X).
Colorblindness
is
due
to
an
X-linked
recessive
gene
as
follows:
*
Normal
female:
X*X*
or
X*XC;
Colorblind
female:
X“XC
e
Normal
male:
X*Y;
Colorblind
male:
X°Y
*
Normal
Klinefelter's:
X*X*Y
or
X*X°Y;
Colorblind
Klinefelter’'s:
X¢X°Y
*
Normal
Turner’s:
X;
Colorblind
Turner’s:
X¢
a.
If
a
colorblind
woman
(X®X°)
and
a
man
with
normal
vision
(X'Y)
produce
a
colorblind
Klinefelter’s
child,
how
can
this
be
explained?
Which
parent
provided
each
sex
chromosome?
b.
If
a
heterozygous
woman
with
normal
vision
(X*X°)
and
a
man
with
normal
vision
(X*Y)
produce
a
colorblind
Turner’s
child,
how
can
this
be
explained?
Which
parent
provided
this
sex
chromosome
and
why
is
there
only
one?
c.
Is
it
possible
for
a
colorblind
male
(X°Y)
and
a
female
that
is
not
colorblind
(X*X*)
to
produce
a
male
child
that
is
chromosomally
normal,
but
colorblind?
Draw
a
Punnett
Square
to
help
you
determine
the
answer.
Among
abnormalities
of
the
sex
chromosomes,
there
are
no
individuals
with
only
a
Y
chromosome
(no
other sex
chromosomes),
but
there
are
individuals
with
only
an
X
chromosome
(Turner's
Syndrome).
a.
Why
are
there
no
instances
of
individuals
with
only
a
Y
chromosome?
b.
Is
this
evidence
for
differences
in
the
genetic
content
between
the X
and
the
Y
chromosome?
Explain
this.
38
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Karyotyping
Worksheet
Group
A
Long
chromosomes;
i
2
3
cEnromers
near
conter.
Giroup
8
Long
o
:hromosomes:
4
5
cenlromere
ot
in
center,
Grroup
C;
Madiom
chromosomes;
centromeres
slighudy
off
center,
6
7
8
9
10
1
12
Group
D¢
Mediom
-
—sn
chromosomes:
13
14
15
captrometes
‘
oear
oo
epd
of
chromosone,
Group
B
Short
‘
w—as
Croup
F:
Shon
chromosomes;
16
17
18
chromosomes;
19
20
contromems
cantromerey
slighdy
off
BEAr
CERLeL.
conier,
Group
G:
Very
shore
Sex
chromosomes:;
21
22
Chromosomes
COnUOmens
near
end,
Smear
#
How
many
chromosomes
does
this
infiividua!
possess?
What
is
the
sex
of
the
individual?
Describe
any
chrormosome
abnormalties
you
have
discovered.
BFBILTIA
%
1998
Flinn
Scientific,
loc.
AN
Right
Reserved,
mmmmummwmmmmm”mmmnm
Karyoryping
Kit.
Cawlog
Mo,
FB11
1L,
from
Flion
Seimmific,
Inc.
Nfimfifmifihfimmrhw
unmummmymmwbrwm,mgmmmlm&mmnufime
reconding,
o
Bny
information
woe
age
and
remieval
sysiom,
without
permisson
in
writing
from
Flina
Seleadfie,
Ine,
39
9/r
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40
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Instructions:
1.
Usc
scissors
to
cul
out
the
individual
chrommosomes,
2,
Match
homologous
pairs
of
chromosomes
and
place
them
on
the
worksheet
in
their
appropriate
space,
Use
the
line
pro-
vided
1o
line
up
the
centromere
region
on
each
chromosome,
Use
the
shape,
size,
banding
patterns,
and
group
descriptions
o
arrange
the
karyotype.
3.
When
your
chromosomes
have
been
classificd
and
you
are
confident
of
their
dentity,
tape
thein
to
your
worksheet
and
answer
the
questions
on
the
worksheet.
BFBIITIA
@
1998
Fline
Scientifiz,
o,
All
Riphts
Reserved.
Reproduction
permasson
18
grantod
omdy
o
sciones
@actmx
who
have
poarchased
the
Humun
Karyoping
Ki,
Caalog
Mo,
FRILE,
from
Flion
Scientific,
Inc.
Mo
purt
of
this
ssteeial
siny
be
reproduced
of
wansowrad
in
any
form
o
by
any
means,
electronic
or
mechanical,
includion,
bl
not
imited
to
pheiocopy,
rroosding,
or
any
infurmation
stor-
ape
and
retrieval
system,
without
permission
in
writing,
from
Hlinn
Scietific,
Tne.
41
.
/
0,
a
i
;
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42
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ww
x4
wemweny
v
v
¢
et
s
s
s
Chromosome
Smear
#2
Instractions:
1.
Use
scissors
to
cut
out
the
individual
chromosomes,
2.
Match
homologous
pairs
of
chromosomes
and
place
themn
on
the
worksheet
in
their
appropriale
space.
Use
the
line
pro-
vided
to
line
up
the
centromere
region
on
each
chromosome.
Use
the
shape,
size,
banding
patierns,
and
group
descriptions
to
arrange
the
karyolype.
3,
When
your
chromosomes
have
been
classified
and
you
are
confident
of
their
identity,
tape
them
to
your
worksheet
and
answer
the
questions
on
the
worksheet.
BFRITIIA
A
160W
1
Scicnofic,
he,
Al
Rights
Reserecd,
Reproduniion
permission
i
gromted
onby
b0
scionce
lechers
who
have
parchased
the
Bluman
Kacyotyping
Kit,
Cnlalog
te,
¥R
L
foom
Plinn
Sritng,
T
Mo
ppet
of
this
materin)l
may
e
reprodoced
or
tansmiticd
io
any
foom
on
by
any
fostos,
elettoome
of
moehanical,
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ot
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1
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sooordiog.
of
sy
informudion
ghee
\
apeer
sl
reteieval
mystons,
Wibour
pergsion
inowndng
from
Plins
Seientitie,
lne,
{4
}
s
9/
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-
.
£
BMRMM
dAae
Ab
MM
G5
&
€0
MY 41
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WERW
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CWWNMETSX{
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WOV
@
¢
<
A
Chromosome
Smear
#3
Instructions:
1.
Use
scissors
to
cut
out
the
individual
chromosomes.
2.
Match
homologous
pairs
of
chromosomes
and
place
them
on
the
worksheet
in
their
appropriate
space.
Usc
the
ling
pro-
vided
1o
line
up
the
ceniromere
region
on
each
chromosome.
Use
the
shape,
size,
banding
patterns,
and
group
descriptions
w
arrange
the
kKaryotype,
3.
When
your
chromosomes
have
been
classificd
and
you
are
confident
of
their
identity,
tape
them
to
your
worksheet
and
answer
the
questions
on
the
worksheet.
BFBITLIA
€
1B
Plinn
Scientific,
Ine,
AR
Rights
Ruserved,
Repruduction
penmdssion
s
granted
only
to
seience
teschers
who
have
purchased
the
Human
Karyatyping
Kin,
Crtabog,
No.
FBTIT,
from
Plios
Seientific,
Ing,
No
part
of
this
material
may
be:
mproduced
or
iransmitied
in
any
form
or
by
any
means,
clecuonte
or
imechanical,
icluding,
but
not
lisdved
to
photocopy,
recording,
or
any information
stor-
Ape
wid
seWicvak
systiol,
withoot
peamission
m
wWiiking
froam
Blinn
Seientilic,
Ine.
/
45
‘9
/
ff‘
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46
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Chromosome
Smear
#4
Instractions:
1.
Use
scissors
to
cut
out
the
individual
chromosomes,
2.
Match
homologous
pairs
of
chromosomes
and
place
them
on
the
workshect
in
their
appropriate
space.
Use
the
line
pro-
vided
to
line
up
the
centromere
region
on
cach
chromosome.
Use
the
shape,
size,
banding
patterns,
and
group
deseriptions
to
arrange
the
karyolype,
3.
When
your
chromosemes
have
been
classificd
und
you
are
confident
of their
identity,
tape
them
fo
your
worksheet
and
answer
the
questions
on
the
worksheet.
BEBTHA
&
1998
Flion
Sciantific,
1oz,
Al
Rights
Rusaved,
Repesluction
permission
is
gemted
enly
to
science
reachers
who
have
gusehased
the
Hurman
Kayotyping
Kit,
Cudog
Mo,
PR
from
Plinn
Seivotifie,
e,
Mot
of
thig
nqeerial
may
be
meproduced
or
bnusmitied
woany
1o
ne
sty
means,
alectronic
ar
prechimical,
icloding,
bt
s
Hedied
1o
phoweopy,
eecording,
or
any
tonedion
st
e
wnd
petrieval
gyston,
withon
peomission
wowridng
froes
Flian
Sdewitie,
e
47
a
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48
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%
Instructions:
I,
Use
scissors
o
cut
out
the
individual
chromosomes.
2.
Match
homologous
pairs
of
chromosomes
and
place
them
on
the
worksheet
in
their
appropriate
space.
Use
the
ling
pro-
vided
1o
line
up
the
centromere
region
on
cach
chromosome.
Use
the
shape,
size,
banding
patterns,
and
group
descriptions
to
arrange
the
karyotype,
3.
When
your
chromosomes
have
heen
classifled
and
you
are
confident
of
their
identity,
tape
them
to
your
worksheet
and
answer
the
questions
on
the
worksheet,
BFRLIIITA
2%
1998
Flinn
Sciewific,
Ioe,
Al
Rights
Reserved,
Reproduction
permdsgion
ig
gramred
only
10
ssicoct
achers
who
have
purchared
the
Theman
Karyodyping
Kit,
Catalog
No.
FB1111,
fom
HFling
Sateneedio,
g,
No
part
of
thiv
matsrial
may
be
reproduced
or
ransmitted
i
sny
Rerm
o
by
sny
means,
slecteonic
o
machanioal,
including,
but
not
limised
1o
photocopy,
recording,
or
any
informasion
-
et
amd
retrieval
sysiem,
without
pormission
in
writing
from
Fling
Scientific,
Inc,
.
49
9/iy
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50
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Lab
#6
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BIO
115
Mutation
Effects
of
Radiation
A
plant
seed
contains
a
dormant
embryo.
The
zygote
has
already
undergone
repeated
division
and
has
formed
an
embryonic
root
and
stem,
but
growth
has
been
halted
and
will
be
resumed
only
if
and
when
the
seed
encounters
conditions
favorable
for
growth
(like
sufficient
moisture).
Since
the
embryo
is
not
growing,
it
can
withstand
a
very
high
dosage
of
radiation
as
compared
with
animal
embryos.
The
irradiation
of
seeds
may
cause
damage
to
the
DNA
which
may
lead
to
changes
in
the
DNA
sequence
of
the
plant.
These
changes
to
the
DNA
sequence
are
called
mutations.
The
mutations
caused
by
irradiation
of
the
seeds
may
cause
morphological
alterations
in
the plants.
Changes
in
the
shape,
size,
and
viability
of
the
plant
are
common.
Morphological
changes
may
be
either
beneficial
or
detrimental
to
the
seed.
The
same
dose
of
irradiation
can
produce
beneficial
mutations
in
some
seeds
and
detrimental
mutations
in
others.
Some
examples
of
beneficial
mutations
are
resistance
to
diseases
and
abnormal
weather
conditions,
increase
in
rate
of
growth,
or
better
quality
and
quantity
of
the
fruits
produced
by
the
plants.
Some
examples
of
detrimental
mutations
are
loss
of
chlorophyll,
slow
growth,
decrease
in
viability,
or
change
in
color/size
of
flowers.
The
normal
germination
and
growth
height
of
the
seeds
may
be
determined
through
the
use
of
control
seeds,
which
have
not
been
exposed
to
radiation.
You
will
“plant”
seeds
from
one
of
the
following
groups:
e
Control
e
50,000
rads
e
150,000
rads
¢
500,000
rads
*
4,000,000
rads
Procedure:
1.
With
gloves
on,
wet
the
paper
towel
and
put
it
into
the
bottom
of
your
petri dish.
You
should
fill
the
petri
dish
most
of
the
way.
2.
Put
afilter
paper
on
top
of
the
paper
towel.
Put
seeds
of
your
given
group
(5
if
squash;
10
if
radish
or
marigold)
on
top
of
the
filter
paper.
4.
Put
the
top
on
the
petri
dish.
You
should
be
able
to
turn
the
petri
dish
vertically
without
the
seeds
falling.
If
the
seeds
fall,
add
more
damp
paper
towel
to
the
bottom
of
your
petri
dish.
5.
Tape
the
petri
dish
so
that
the
lid
and
bottom
can’t
become
separated
from
one
another.
6.
Label
the
petri
dish
with
your
initials,
your
lab
section,
and
the
radiation
treatment
of
the
seeds
you
planted.
7.
Vertically
place
your
petri
dish
in
a
small
glass
dish
that
contains
a
small
amount
of
water.
This
will
make
sure
that the
seeds
stay
damp
throughout
the
experiment.
8.
For
the
next
three
weeks
in
lab,
you
will
record
the
number
of
seeds
that
have
germinated
in
your
dish,
and
the
average
height
of
the
seeds
that
have
germinated.
You
should
observe
any
other
morphological
changes
that
occurs
in
your
seeds.
9.
After
the
experiment,
you
will
make
graphs
that
show
the
growth
and
germination
of
the
seeds
over
time.
This
will
allow
you
to
examine
the
effect
of
radiation
on
plant
growth.
w
52
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l
|
Day
7
seeds
number
germinated
‘average
height
(cm)
control
50,000
rads
150,000
rads
500,000
rads
4,000,000
rads
Day
14
seeds
number
germinated
average
height
(cm)
control
50,000
rads
150,000
rads
500,000
rads
4,000,000
rads
number
germinated
average
height
(cm)
SOQ,GOO
rads
4,000,000
rads
53
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For
each
group
of
seeds,
plot
the
height
(vertical
axis)
against
time
in
days
(horizontal
axis).
Be
sure
to
use
different
symbols
for
each
of
the
different
groups
L
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.
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(cm)
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e
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«
-
Time
(days)
54
4
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L
‘
t,
|
4
!
Number
-l
!
Germinated
'
l
i
ey
¢
«
-
Time
(days)
55
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Mutation
Effects
of
Radiation
Lab
Questions
1.
Make
two
line
graphs, one
for
height
of
plants
and
one
for
number
of
plants
germinated.
Make
sure
to
label
all
axes
with
units.
You
may
draw
the
graphs
and
take
a
picture
or
make
the
graphs
on
the
computer
with
excel/sheets,
but
either
way
please
put
the
graphs
in
your
final
word
doc
submission.
a.
Height:
For
each
group
of
seeds,
plot
the
height
(vertical
axis)
against
time
in
days
(horizontal
axis).
Each
seed
group
(different
rad
levels)
should
be
a
different
line.
b.
Number
germinated:
For
each
group
of
seeds,
plot
the
number
germinated
(vertical
axis)
against
time
in
days
(horizontal
axis).
Each
seed
group
(different
rad
levels)
should
be
a
different
line.
2.
Is
there
a
correlation
(positive
or
negative)
between
the
number
of
germinated
seeds
and
amount
of
radiation
administered?
Explain
this.
3.
Is
there
a
correlation
(positive
or
negative)
between
the
average
plant
heights
and
amount
of
radiation
administered?
Explain
this.
4.
What
can
be
concluded
concerning
the
effects
of
radiation
on
seed
germination?
Is
radiation
beneficial
or
harmful
to
the
seeds?
5.
What
can
be
concluded
concerning
the
effects
of
radiation
on
plant
height?
Is
radiation
beneficial
or
harmful
to
the
seeds?
6.
Which
treatment
group
did
you
expect
to
have
the
most
abnormalities
in
comparison
to
the
control
group?
Why?
Did
you see
this?
7.
What
are
two
possible
positive
abnormalities
that
could
occur
in
plants
from
exposure
to
strong
radiation?
8.
What
are
two
possible
negative
abnormalities
that
could
occur
in
plants
from
exposure
to
strong
radiation?
9.
How
does
strong
radiation
cause
abnormalities
in
plants?
10.
Why
are
control
groups
needed
in
this
experiment?
56
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Lab
#7
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DNA PATERNITY
TESTING
SIMULATION
EDVO-Kit
114
Background
Information
DNA
fingerprinting
(also
called
DNA
typing)
allows
4
for
the
identification
of
the
source
of
DNA
samples.
The
method
has
become
very
important
to
provide
evidence
in
paternity
and
criminal
cases.
In
contrast
to
the
more
conventional
methodologies,
such
as
Decreasing
blood
typing,
which
can
only
exclude
a
suspect,
DNA
Fragment
fingerprinting
can
provide
positive
identification
Size
with
great
accuracy.
‘
Paternity
determination
based
on
DNA
analysis
(genetic
DNA
fingerprinting)
has
become
an
impor-
tane
;
g/p]qltdher
tant
procedure
for
matching
children
with
biological
ng:
3
Fatlher
fathers
and
mothers.
Examples
of
recent
court
cases
lane
4
Unrelated
that
have
utilized
this
procedure
have
included
rape,
incest,
immigration,
citizenship
of
children
to
the
United
States
and
matching
of
children
with
parents
S
.
who
were
mismatched
at
birth
due
to
hospital
er-
The
child's
(lane
2)
DNA
pattern
contains
DNA
from
rors.
This
type
of
testing
is
also
used
during
unrest
the
mother
(lane
1)
and
the
biological
father
(lane
3).
as
in
nations
in
civil
war
where
children
are
often
_J
separated
from
parents
and
subsequently
reunited.
Figure
1:
For
paternity
DNA
fingerprinting,
samples
obtained
from
the
mother,
the
child,
and
possible
fathers
are
analyzed.
A
child's
DNA
is
a
composite
of
its
parent
DNAs.
There-
fore,
comparison
of
DNA
fragmentation
patterns
obtained
from
the
mother
and
child
will
give
a
partial
match.
Bands
in
the
child's
DNA
fingerprint
that
are
not
present
in
the
mother's
must
be
contributed
by
the
father.
Because
of
allelic
differences,
the
DNA
bands
present
in
the
child's
fingerprint
must
be
found
in
either
the
father's
or
mother's
fingerprint.
Prior
to
the
advent
of
the
Polymerase
Chain
Reaction
(PCR),
DNA
fingerprinting
involved
the
electrophoretic
analysis
of
DNA
fragment
sizes
generated
by
restriction
enzymes
followed
by
Southern
Blot
Analysis.
Restriction
enzymes
are
endonucleases
which
catalyze
the
cleavage
of
the
phosphate
bonds
within
both
strands
of
DNA.
They
require
Mg+2
for
activity
and
generate
a
5
prime
(5')
phosphate
and
a
3
prime
(3')
hydroxyl
group
at
the
point
of
cleavage.
The
distinguishing
feature
of
restriction
enzymes
is
that
they
only
cut
at
very
specific
sequences
of
bases
called
recognition
sites.
Restriction
enzymes
are
produced
by
many
different
species
of
bacteria
(includ-
ing
blue-green
algae).
Over
3,000
restriction
enzymes
have
been
discovered
and
catalogued.
Restriction
enzymes
are
named
according
to
the
organism
from
which
they
are
isolated.
This
is
done
by
using
the
first
letter
of
the
genus
followed
by
the
first
two
letters
of
the
species.
Only
certain
strains
or
substrains
of
a
particular
species
may
be
a
producer
of
restriction
enzymes.
The
type
of
strain
or
substrain
sometimes
follows
the
species
designation
in
the
name.
1.800.£DVOTEK
»
Fax
202.370.1501
+
info®edvotek.com
*
www.edvotek.com
E
DVOT
E
K®
Duplication
of
any
part
of
this
document
is
permitted
for
non-profit
educational
purposes
only.
Copyright
©
1989-2014
EDVOTEK,
Inc.,
all
rights
reserved.
114.141217
4
58
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EDVO-Kit
114
DNA PATERNITY
TESTING
SIMULATION
Finally,
a
Roman
numeral
is
always
used
to
designate
one
out
of
possibly
several
dif-
ferent
restriction
enzymes
produced
by
the
same
organism
or
by
different
substrains
of
the
same
strain.
Restriction
enzymes
recognize
specific
double
stranded
sequences
in
DNA.
Most
rec-
ognition
sites
are
4
to
8
base
pairs
in
length.
Cleavage
occurs
within
or
near
the
site.
The
cleavage
positions
are
indicated
by
arrows.
With
some
exceptions,
recognition
sites
are
frequently
symmetrical,
i.e.,
both
DNA
strands
in
the
site
have
the
same
base
sequence
when
read
5'
to
3'.
Such
sequences
are
called
palindromes.
It
is
these
sites
in
DNA
that
are
substrates
for
restriction
enzymes.
In
DNA
paternity
and
fingerprint-
ing
laboratories,
the
commonly
used
restriction
enzymes
are
Hae
Ill
(GG'CC)
and
Hinf
|
(G'ANTC),
which
are
4-base
and
5-base
cutting
enzymes
respectively.
In
this
experiment,
the
DNAs
from
a
hypothetical
paternity
case
are
cut
by
a
restriction
enzyme,
which
is
a
six-base
cutting
enzyme.
Examples
of
six-base
cutting
enzymes
include
Bam
HI
and
Pst
I.
The
recognition
sites
for
these
restriction
enzymes
are:
Pst
|
BamHl
..
lccTAGlG|
5
stl
5
|cAceTC|
5
The
size
of
the
DNA
fragments
generated
by
restriction
enzyme
cleavage
depends
on
the
distance
between
the
recognition
sites.
No
two
individuals
have
exactly
the
same
pattern
of
restriction
enzyme
recognition
sites.
There
are
several
reasons
for
this
fact.
A
large
number
of
alleles
exist
in
the
population.
Alleles
are
alternate
forms
of
a
gene.
It
is
estimated
that
about
25%
of
all
human
genes
occur
in
mul-
tiple
alleles
which
are
called
polymorphisms.
Alleles
result
in
alternative
expressions
of
genetic
traits
which
can
be
dominant
or
recessive
and
are
inherited
in
a
Mendelian
pattern
just
as
genes.
Restriction
Organism
Enzyme
Chromosomes
occur
in
matching
pairs,
one
of
maternal
and
the
Bam
Hi
Bacillus
omyloliquetaciens
other
of
paternal
origin.
The
two
copies
of
a
gene
(which
can
:
i
Hae
Il
Haemophilus
aegyplius
be
alleles)
at
a
given
chromosomal
locus,
and
which
represent
p
gyp
a
composite
of
the
parental
genes,
constitutes
the
unique
gen-
Eco
Rl
Eccherichia
coli.
strain
RY13
otype
for
an
offspring.
It
follows
that
alleles
have
differences
’
in
their !)age
sequences
which
c_onsequentily
Creates
differences
Hinf]
Haemophilus
influenzae
Rf
in
the
distribution
and
frequencies
of
restriction
enzyme
rec-
ognition
sites.
Other
differences
in
base
sequences
between
individuals
can
occur
because
of
mutations
and
deletions.
Such
Figure
2:
changes
can
also
create
or
eliminate
a
recognition
site.
Restriction
enzyme
names
and
bacterial
sources.
_J
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DNA PATERNITY
TESTING
SIMULATION
EDVO-Kit
114
The
example
in
Figure
3
shows
how
a
silent
mutation
can
eliminate
a
recogni-
tion
site
but
leave
a
protein
product
unchanged.
Individual
variations
in
the
distances
between
recognition
sites
in
chromosomal
DNA
are
often
caused
by
(
Coding
DNA
Codon
changed
Eco
Rlsite
Mutation
by
mutation
5
—ACGAATTCC—
3'
oy
5"
—
ACG
AACTCC—
3'
\/
intervening
repetitive
base
sequences.
Repetitious
sequences
constitute
a
large
fraction
of
the
mammalian
genome
and
have
no
known
genetic
function.
These
sequences
can
occur
between
genes
or
are
adjacent
to
them.
They
are
also
found
within
introns.
Ten
to
fifteen
percent
of
mammalian
DNA
consists
of
sets
of
repeated,
short
sequences
of
bases
that
are
tandemly
arranged
in
arrays.
The
length
of
these
arrays
(the
amount
of
repeated
sets)
varies
between
individuals
at
different
chromosomal
loci.
Protein
Product
H,N
—
Thr
-
Asn
-
Ser
—
COOH
Protein
Product
H,N
—
Thr
-
Asn
-
Ser
—COOH
Figure
3:
Effect
of
silent
mutation
on
protein.
J
TGTTTA
|
TGTTTA
|
TGTTTA
|
.........
variable
number
When
these
arrays
are
flanked
by
recognition
sites,
the
length
of
the
repeat
will
determine
the
size
of
the
restriction
enzyme
fragment
generated.
Variations
in
the
length
of
these
fragments
between
different
individuals,
in
a
population,
are
known
as
restriction
fragment
length
polymorphisms,
RFLPs.
Several
hundred
RFLPs
have
been
mapped
on
all
23
chromosomes.
RFLPs
are
a
manifestation
of
the
unique
molecular
genetic
profile,
or
“fingerprint”,
of
an
individual’s
DNA.
As
shown
in
Figure
4,
there
are
several
types
of
these
short,
repetitive
sequences
that
have
been
cloned
and
purified.
In
Southern
blot
analysis,
DNA
probes
are
used
to
detect
the
length
differences
between
these
repetitive
sequences.
DNA
probes
are
short
fragments
of
single
stranded
DNA
that
are
isotopically
or
non-isotopically
labeled.
DNA
probes
will
complement
and
hybridize
(attach)
to
single
stranded
DNA.
Southern
blot
analysis
requires
electrophoresis,
denaturation
of
the
DNA
fragments,
transfer
of
DNA
to
a
membrane,
and
exposure
to
probes
to
detect
DNA
Fingerprints.
(
Allele
1
Allele
2
I
[y
]
Restriction
larger
Fragments
|
[T
1
smaller
\l,
=
recognition
site
=T
=
repetitious
sequence
array
(variable length)
Figure
4:
Restriction
fragment
length
polymorphisms..
J
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¢
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EDVOTEK,
Duplication
of
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part
of
this
document
is
permitted
for
non-profit
educational
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only.
Copyright
©
1989-2014
EDVOTEK,
Inc.,
all
rights
reserved.
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EDVO-Kit
114
DNA PATERNITY
TESTING
SIMULATION
There
are
two
types
of
probes
commonly
used
for
genetic
identification.
The
single-locus
probes
(SLPs)
which
detect
a
single
segment
of
the
repetitive
DNA
located
at
a
specific
site
on
a
single
chromosome.
This
will
result
in
one
or
two
DNA
bands
corresponding
to
one
or
both
chromosome
segments
recognized.
If
the
segments
on
the
chromosome
pairs
are
the
same,
then
there
will
be
one
band.
On
the
other
hand,
if
they
are
different,
it
will
appear
as
two
bands.
Several
SLPs
are
available
and
are
used
less
frequently
since
more
than
one
person
can
exhibit
the
same
exact
pattern
for
a
specific
SLP.
Multiple-
Locus
probes
(MLPs)
detect
multiple
repetitive
DNA
segments
located
on
many
chromo-
somes
yielding
20-30
bands.
Because
of
the
multi-band
patterns,
the
chances
of
two
people
chosen
at
random
having
the
same
pattern
is
enormously
remote.
For
example,
it
is
calculated
that
two
unrelated
individuals
having
the
identical
DNA
pattern
detected
by
MLPs
as
an
average
is
1
in
30
billion.
It
should
be
kept
in
mind
that
the
total
human
population
on
earth
is
between
5-6
billion.
Currently,
the
polymerase
chain
reaction
(PCR)
is
routinely
used
in
forensics
to
analyze
DNA
(Figure
4).
This
technique
requires
about 500-fold
less
DNA
than
Southern
blot
RFLP
analysis
and
is
less
time-consuming.
PCR
amplification
(Figure
5)
uses
an
enzyme
known
as
Taq
DNA
polymerase.
This
enzyme,
originally
was
purified
from
a
bacterium
that
in-
habits
hot
springs
and
is
stable
at
very
high
(near
boiling)
temperatures.
Also
included
in
the
PCR
reaction
mixture
are
two
synthetic
oligonucleotides
known
as
“primers”
and
the
extracted
DNA.
The
region
of
DNA
to
be
amplified
is
known
as
the
“target”.
In
the
first
step
of
the
PCR
reaction,
the
template
complementary
DNA
strands
are
sepa-
rated
(denatured)
from
each
other
at
94°C,
while
the Taq
polymerase
remains
stable.
In
the
second
step,
known
as
annealing,
the
sample
is
cooled
to
40°-65°C,
to
allow
hybrid-
ization
of
the
two
primers,
one
to
each
of
the
two
strands
of
the
template
DNA.
In
the
third
step,
known
as
extension,
the
temperature
is
raised
to
72°C
and
the
Taq
polymerase
adds
nucleotides
to
the
primers
to
synthesize
the
new
complementary
strands.
These
three
steps
-
denaturation,
annealing,
and
extension
-
constitute
one
PCR
“cycle”.
This
process
is
typically
repeated
for
20-40
cycles,
amplifying
the
target
sequence
within
DNA
exponentially
(Figure
5).
PCR
is
performed
in
a
thermal
cycler,
an
instrument
that
is
programmed
to
rapidly
heat,
cool
and
maintain
samples
at
designated
temperatures
for
varying
amounts
of
time.
The
PCR
products
are
separated
by
agarose
gel
electrophoresis
and
DNA
fingerprints
are
analyzed.
In
forensics
and
DNA
paternity
testing,
PCR
is
used
to
amplify
and
examine
highly
vari-
able
(polymorphic)
DNA
regions.
These
are
regions
that
vary
in
length
from
individual
to
individual
and
fall
into
two
categories:
1)
variable
number
of
tandem
repeats
(VNTR)
and
2)
STR
(short
tandem
repeats).
A
VNTR
is
a
region
that
is
variably
composed
of
a
15-70
base
pair
sequence,
typically
repeated
5-100
times.
An
STR
is
similar
to
a
VNTR
except
that
the
repeated
unit
is
only
2-4
nucleotides
in
length.
By
examining
several
different
VNTRs
or
STRs
from
the
same
individual,
investigators
obtain
a
unique
DNA
profile
for
that
individual
which
is
unlike
that
of
any
other
person
(except
for
identical
twins).
In
this
simulation
experiment,
DNA
was
extracted
from
samples
obtained
from
the
mother,
child
and
two
possible
fathers.
The
objective
is
to
analyze
and
match
the
DNA
fragment
patterns
after
agarose
gel
electrophoresis
and
determine
if
Father
1
or
Father
2
is
the
biological
parent
of
the
child.
THIS
EXPERIMENT
DOES
NOT
CONTAIN
HUMAN
DNA.
1.800.EDVOTEK
©
Fax
202.370.1501
-
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Duplication
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any
part
of
this
document
is
permitted
for
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educational
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only.
Copyright
©
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DVOT
E
K®
EDVOTEK,
Inc.,
all
rights
reserved.
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DNA PATERNITY
TESTING
SIMULATION
EDVO-Kit
114
Target
Sequence
5’
WM
M
3
Separation
of
3
MWV
AW
5
two
DNA
strands
*
=
Primer
1
11
-
|
n
=
Primer
2
5'
'
/wameeee—
AW
3
¢
Denature
94°C
Cycle
1
¢
>
j
Anneal
‘
\
5'—
,
2
primers
|
[3'mw——————
1
5
40°C
-
65°C
5
—
5’
AV
—
A
3]
<_}
Extension
3!
5'
e
5
72°C
i
|
Cycle
3
Figure
5:
DNA
Amplification
by
the
Polymerase
Chain
Reaction
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is
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DNA PATERNITY
TESTING
SIMULATION
EDVO-Kit
114
Experiment
Overview
Prepare
agarose
gel
in
casting
tray
Remove
end
blocks
&
comb,
then
submerge
gel
under
buffer
in
electrophoresis
chamber
@
Load
each
sample
in
consecutive
wells
Attach
safety
cover,connect
leads
to
power
source
and
conduct
electrophoresis
@
After
electrophoresis,
transfer
gel
for
staining
(£D)
InstaStain®
Blue
123
456
or
FlashBlue™
@
=
DNA
stain.
Analysis
on
white
light
source.
(+>
Gel
pattern
will
vary
depending
upon
experiment.
1.800.£DVOTEK
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E
DVOT
E
K®
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of
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part
of
this
document
is
permitted
for
non-profit
educational
purposes
only.
Copyright
©
1989-2014
EDVOTEK,
Inc.,
all
rights
reserved.
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EDVO-Kit
114
DNA
PATERNITY
TESTING
SIMULATION
Module
I:
Agarose
Gel
Electrophoresis
Concentrated
'
;
‘
Distilled
buffer
water
~
IMPORTANT:
If
you
are
unfamiliar
with
agarose
gel
prep
and
electrophoresis,
detailed
instructions
and
helpful
resources
are
available
at
www.edvotek.com
Wear
gloves
CASTING
THE
AGAROSE
GEL
and
safety
goggles
J
1.
DILUTE
concentrated
50X
Electrophoresis
buffer
with
distilled
water
(refer
to
Table
A
for
correct
volumes
depending
on
the
size
of
your
gel
casting
tray).
2.
MIX
agarose
powder
with
buffer
solution
in
@
250
ml flask
(refer
to
Table
A).
3.
DISSOLVE
agarose
powder
by
boiling
the
solution.
MICROWAVE
the
solution
on
high
for
1
minute.
Care-
fully
REMOVE
the
flask
from
the
microwave
and
MIX
by
swirling
the
flask.
Continue
to
HEAT
the
solution
in
15-second
bursts
until
the
agarose
is
completely
dissolved
(the
solution
should
be
clear
like
water).
4.
COOL
agarose
to
60°
C
with
careful
swirling
to
promote
even
dissipation
of
heat.
5.
While
agarose
is
cooling,
SEAL
the
ends
of
the
gel-casting
tray
with
the
rubber
end
caps.
PLACE
the
well
template
(comb)
in
the
appropriate
notch.
6.
POUR
the
cooled
agarose
solution
into
the
pre-
pared
gel-casting
tray.
The
gel
should
thoroughly
solidify
within
20
minutes.
The
gel
will
stiffen
and
become
less
transparent
as
it
solidifies.
7.
REMOVE
end
caps
and
comb.
Take
particular
care
when
removing
the
comb
to
prevent
damage
to
the
wells.
Table
A
Individual
0.8%
UltraSpec-Agarose™
Gel
Size
of
Gel
Concentrated
Distilled
Amt
of
TOTAL
Casting
tray
|
Buffer
(50x)
+
Water
+
Agarose
=
Volume
T
x7cem
0.6
ml
29.4m
0.23
4
30
ml
7x10
cm
1.0
mi
49.0m!
0.39
¢
50
ml
Tx14
cm
1.2
ml
58.8ml
0.46
¢
60
ml
1.800.£EDVOTEK
»
Fax
202.370.1501
-
info@edvotek.com
www.edvotek.com
Duplication
of
any
part
of
this
document
is
permitted
for
non-profit
educational
purposes
only.
Copyright
©
1989-2014
E
DVOT
E
K®
EDVOTEK,
Inc.,
all
rights
reserved.
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DNA
PATERNITY
TESTING
SIMULATION
12
Module
I:
Agarose
Gel
Electrophoresis
|
Po:r
lru——l'
1X
Diluted
Buffer
EDVO-Kit
114
\_
Includes
EDVOTEK’s
All-NEW
DNA
Standard
Marker
-
Better
separation
-
Easier
band
measurements
-
No
unused
bands
NEW
DNA
Standard
ladder
sizes:
6751,
3652,
2827,
1568, 1118,
825,
630
.
(€
10.
11.
<=
RUNNING
THE
GEL
Wear
gloves
and
safety
goggles
y
-
REMINDER:
Before
loading
the
samples,
make
sure
the
gel
is
properly
oriented
in
the
ap-
paratus
chamber.
J
8.
PLACE
the
gel
(still
on
the
tray)
into
the
electrophoresis
chamber.
COVER
the
gel
with
1X
Electrophoresis
Buffer
(See
Table
B
for
reccommended
volumes).
The
gel
should
be
completely
submerged.
9.
PUNCTURE
the
foil
overlay
of
the
QuickStrip™
with
a
pipet
tip.
LOAD
the
entire
sample
(35
pl)
into
the
well
in
the
order
indicated
by
Table
1,
at
right.
10.
PLACE
safety
cover
on
the
unit.
CHECK
that
the
gel
is
prop-
erly
oriented.
Remember,
the
DNA
samples
will
migrate
_
toward
the
positive
(red)
electrode.
esla
8
Gl
benceting
11.
CONNECT
leads
to
the
power
source
and
PERFORM
elec-
Lane
1
|
TubeA
|
Standard
DNA
Marker
trophoresis
(See
Table
C
for
time
and
voltage
guidelines).
2
|
TubeB
|
Mother
DNA
cut
with
Enzyme
AIIcl)lw
the
tracking
dye
to
migrate
at
least
3.5
cm
from
the
3
|
TubeC
|
Child
DNA
cut
with
Enzyme
wells.
.
4
Tube
D
Father
1
DNA
cut
with
E
12.
After
electrophoresis
is
complete,
REMOVE
the
gel
and
cast-
o
e
-
W!
nayme
ing
tray
from
the
electrophoresis
chamber
and
proceed
to
)
|
TUbE
Father
2
DNA
cut
with
Enzyme
instructions
for
STAINING
the
agarose
gel.
Table
Table
B
1x
£lectrophoresis
Buffer
(Chamber
Buffer)
C
Time
&
Voltage
Guidelines
(0.8%
Agarose
Gel)
Dilution
Electrophoresis
Model
.
£DVOTEK
Total
Volume
e
M12
(classic)
Model
Required
Sg:
f;
Fo:rc'
?,3:}:::‘
MG+
M12
(new)
&
M36
Volts
Min.
/
Max,
Min.
/
Max,
Min.
/
Max,
ME+
&
M12
(new)
|
300
ml
6
ml
R
150
|
15/20
min.
|
20/30
min.
|
25/
35
min.
M12
(classic)
400
ml
8
ml
392
ml
125
|
20/30
min.
|
30/35
min.
|
35745
min.
M36
1000
ml
20
ml
980
ml
75
35745
min.
|
55/70
min.
60
7/
90
min.
1.800.EDVOTEK
©
Fax
202.370.1501
¢«
info@®edvotek.com
°
www.edvotek.com
EDVOTEK,
EDVOTEK,
Inc.,
all
rights
reserved.
114.141217
65
Duplication
of
any
part
of
this
document
is
permitted
for
non-profit
educational
purposes
only.
Copyright
©
1989-2014
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EDVO-Kit
114
DNA
PATERNITY
TESTING
SIMULATION
Module
lI-A:
Staining
Agarose
Gels
Using
FlashBlue™
“a,"
2
.
||
Concentrated
fi
Distilled
FlashBlue™
Stain
water
Wear
gloves
Flask
and
safety
gogglesJ
3.
4
ol
Pour
1.
DILUTE
10
ml
of
10x
concentrated
FlashBlue™
with
90
ml
of
water
in
a
flask
and
MIX
well.
2.
REMOVE
the
agarose
gel
and
casting
tray
from
the
electrophoresis
chamber.
SLIDE
the
gel
off
of
the
cast-
ing
tray
into
a
small,
clean
gel-staining
tray.
3.
COVER
the
gel
with
the
1x
FlashBlue™
stain
solution.
STAIN
the
gel
for
5
minutes.
For
best
results,
use
an
orbital
shaker
to
gently
agitate
the
gel
while
staining.
STAINING
THE
GEL
FOR
LONGER
THAN
5
MINUTES
WILL
REQUIRE
EXTRA
DESTAINING
TIME.
4.
TRANSFER
the
gel
to
a
second
small
tray.
COVER
the
gel
with
water.
DESTAIN
for
at
least
20
minutes
with
gentle
shaking
(longer
periods
will
yield
better
results).
Frequent
changes
of
the
water
will
acceler-
ate
destaining.
5.
Carefully
REMOVE
the
gel
from
the
destaining
liquid.
VISUALIZE
results
using
a
white
light
visualization
system.
DNA
will
appear
as
dark
blue
bands
on
a
light
blue
background.
ALTERNATIVE
PROTOCOL:
1.
DILUTE
one
ml
of
concentrated
FlashBlue™
stain
with
149
ml
dH,O0.
2.
COVER
the
gel
with
diluted
FlashBlue™
stain.
3.
SOAK
the
gel
in
the
staining
liquid
for
at
least
three
hours.
For
best
results,
stain
gels
overnight.
1.800.£DVOTEK
»
Fax
202.370.1501
«
info@edvotek.com
*
www.edvotek.com
Duplication
of
any
part
of
this
document
is
permitted
for
non-profit
educational
purposes
only.
Copyright
©
1989-2014
E
DVOT
E
K®
EDVOTEK,
Inc.,
all
rights
reserved.
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DNA PATERNITY
TESTING
SIMULATION
EDVO-Kit
114
Module
lI-B:
Staining
Agarose
Gels
Using
InstaStain®
Blue
1.
2.
3.
.
q,
7
=
5
6.
7.
8
A
%75
wl
=
DESTAIN
==
..+
O
1.
Carefully
REMOVE
the
agarose
gel
and
casting
tray
from
the
electrophoresis
chamber.
SLIDE
the
gel
off
of
the
casting
tray
on
to
a
piece
of
plastic
wrap
on
a
flat
surface.
2.
MOISTEN
the
gel
with
a
few
drops
of
electrophoresis
buffer.
3.
Wearing
gloves,
PLACE
the
blue
side
of
the
InstaStain®
Blue
card
on
the
gel.
4.
With
a
gloved
hand,
REMOVE
air
bubbles
between
the
card
and
the
gel
by
firmly
run-
Wear
gloves
ning
your
fingers
over
the
entire
surface.
Otherwise,
those
regions
will
not
stain.
and
safety
9099'95J
5.
PLACE
the
casting
tray
on
top
of
the
gel/card
stack.
PLACE
a
small
weight
(i.e.
an
-
empty
glass
beaker)
on
top
of
the
casting
tray.
This
ensures
that
the
InstaStain®
Blue
NOTE:
card
is
in
direct
contact
with
the
gel
surface.
STAIN
the
gel
for
10
minutes.
DO
NOT
STAIN
6.
REMOVE
the
InstaStain®
Blue
card.
If
the
color
of
the
gel
appears
very
light,
reapply
GELS
IN
THE
the
Instastain®
Blue
card
to
the
gel
for
an
additional
five
minutes.
ELECTROPHORESIS
7.
TRANSFER
the
gel
to
a
small,
clean
gel-staining
tray.
COVER
the
gel
with
about
75
mL
APPARATUS.
p
of
distilled
water
and
DESTAIN
for
at
least
20
minutes.
For
best
results,
use
an
orbital
shaker
to
gently
agitate
the
gel
while
staining.
To
accelerate
destaining,
warm
the
distilled
water
to
37°C
and
change
it
frequently.
8.
Carefully
REMOVE
the
gel
from
the
destaining
liquid.
VISUALIZE
results
using
a
white
light
visualization
system.
DNA
will
appear
as
dark
blue
bands
on
a
light
blue
background.
ALTERNATIVE
PROTOCOL:
1.
Carefully
SLIDE
the
agarose
gel
from
its
casting
tray
into
a
small,
clean
tray
containing
about
75
ml
of
dis-
tilled/deionized
water
or
used
electrophoresis
buffer.
The
gel
should
be
completely
submerged.
2.
Gently
FLOAT
the
InstaStain®
Blue
card(s)
on
top
of
the
liquid
with
the
stain
(blue
side)
facing
toward
the
gel.
Each
InstaStain®
Blue
card
will
stain
49
cm2
of
gel
(7
x
7
cm).
3.
COVER
the
tray
with
plastic
wrap
to
prevent
evaporation.
SOAK
the
gel
in
the
staining
liquid
for
at
least
3
hours.
The
gel
can
remain
in
the
liquid
overnight
if
necessary.
4.
Carefully
REMOVE
the
gel
from
the
staining
tray.
VISUALIZE
results
using
a
white
light
visualization
system.
DNA
will
appear
as
dark
blue
bands
on
a
light
blue
background.
1.800.£DVOTEK
»
Fax
202.370.1501
+
info®edvotek.com
*
www.edvotek.com
E
DVOT
E
K®
Duplication
of
any
part
of
this
document
is
permitted
for
non-profit
educational
purposes
only.
Copyright
©
1989-2014
EDVOTEK,
Inc.,
all
rights
reserved.
114.141217
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Paternity
Testing
Lab
Questions
Include
a
picture
of
your
gel
along
with
the
answers
to
the
following
questions.
Some
of
the
bands
in
the
gel
may
be
faint,
but
you
should
be
able
to
see
enough
of
them
to
give
you
the
information
that
you
need
for
the
following
questions.
Increasing
the
brightness
or
contrast
of
pictures
while
looking
at
the
gels
may
help.
1.
9.
Label
the
gel
with
the
following:
Lane/well
number.
The
tube
letter
that
is
in
each
well
and
what
each
sample
is.
The
positive
and
negative
ends
of
the
gel.
The
sizes
of
the
standard
marker
fragments.
The
known
sizes
are
6751,
3652,
2827,
1568,
1118,
825,
and
630
base
pairs.
You
may
not
be
able
to
see
all
of
the
bands,
so
refer
to
the
picture
below.
Indicate
which
size
corresponds
to
each
of
the
bands
in
your
marker
lane.
e.
The
approximate
sizes
of
the
bands
that
you
can
see
in
each
sample.
Do
this
by
comparing
the
bands
in
each
sample
to
the
standard
marker
fragments.
L
S
What
will
travel
farther
in
a
gel,
small
or
large
fragments
of
DNA?
Is
DNA
positively
or
negatively
charged?
What
are
restriction
enzymes?
What
is
a
RFLP
and
why
does
it
cause
bands
of
different
sizes?
What
function
do
repetitive
base
sequences
have
in
DNA?
What
percentage
of
the
mammalian
genome
do
they
make
up?
Could
two
people
ever
have
the
same
DNA
fingerprint?
Why
or
why
not?
Which
potential
father
is
the
actual
father
of
the
child?
Explain
how
you
know
this.
Why
is
the
unrelated
male
not
the
father
of
the
child?
10.
What
is
DNA
fingerprinting
used
for
other
than
paternity
testing?
\-
Includes
EDVOTEK's
All-NEW
\
DNA
Standard
Marker
+
Better
separation
+
Easier
band
measurements
«
No
unused
bands
NEW
DNA
Standard
ladder
sizes:
6751,
3652, 2827,
1568, 1118,
825,
630
|
i
e
68
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Lab
#8
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NUCLEIC
ACID
TESTING
FOR
COVID-19
EDVO-Kit
#123
Background
Information
Each
year,
experts
estimate
that
coronaviruses
cause
15-30%
of
(
IMPORTANT
DEFINITIONS:
all
common
cold
cases.
These
symptoms
are
generally
mild
and
include
fever
and
sore
throat.
Sometimes
a
novel
strain
of
the
vi-
rus
emerges
that
causes
severe
respiratory
distress
(for
example
SARS
in
2003
and
MERS
in
2012).
SARS-CoV-2
is
a
novel
coronavi-
rus
that
has
caused
a
worldwide
outbreak
of
respiratory
disease.
COVID-19,
or
Coronavirus
Disease
2019:
This
is
the
The
first
cases
of
COVID-19
were
diagnosed
in
December
2019
disease
caused
by
SARS-CoV-2,
characterized
by
fever,
and
traced
to
the
emergence
of
the
SARS-CoV-2
virus.
According
cough,
and
shortness
of
breath.
SARS-CoV-2,
or
Severe
Acute
Respiratory
Syndrome
Coronavirus
2:
The
name
of
the
novel
coronavirus
responsible
for
the
current
pandemic.
to
the
World
Health
Organization,
COVID-19
spread
worldwide
in
Outbreak:
A
rapid
increase
in
the
number
of
cases
of
a
very
short
period
of
time.
Public
health
officials
are
currently
a
disease
at
a
specific
time
and
place
working
on
strategies
to
identify
infected
individuals
and
to
pre-
Pandemic:
An
outbreak
of
a
disease
in
many
different
vent
the
further
spread
of
the
virus.
geographic
areas
that
affects
a
significant
proportion
of
the
population.
/
Figure
1:
Coronavirus
structure.
Coronaviruses
have
a
single-stranded
RNA
genome
wrapped
in
a
helical
capsid.
A
host-derived
membrane
envelope
surrounds
the
capsid.
The
envelope
is
studded
with
proteins
that
help
the
virus
infect
cells.
By
electron
microscopy,
the
envelope
proteins
create
a
hazy
halo
around
the
virus
particle.
Scientists
described
them
with
the
Latin
word
corona,
which
means
“crown”
or
“halo”
(Figure
1).
Like
all
coronaviruses,
SARS-CoV-2
has
four
main
structural
proteins.
Monomers
of
the
nucleocapsid
protein
(N)
link
to-
gether
to
form
a
helical
capsid
which
wraps
around
and protects
the
RNA
genome.
Embedded
in
the
membrane
are
several
viral
proteins:
the
spike
(S),
the
envelope
(E)
and
the
membrane
(M)
proteins.
The
S
protein
binds
with
human
cell
surface
proteins,
allowing
the
virus
to
inject
its
genetic
material
into
its
host
cells.
The
M
protein
coordinates
interactions
between
the
other
viral
proteins
and
the
host
cell
factors,
turning
cells
into
virus
factories.
As
a
viroporin,
the
E
protein
binds
to
itself
to
form
channels
that
facilitate
viral
release.
1.800.EDVOTEK
©
Fax
202.370.1501
-
info@edvotek.com
-
www.edvotek.com
E
DVOT
E
K®
Duplication
of
any
part
of
this
document
is
permitted
for
non-profit
educational
purposes
only.
Copyright
©
1989-2021
EDVOTEK,
Inc.,,
all
rights
reserved.
123.200824
70
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EDVO-Kit
#123
NUCLEIC
ACID
TESTING
FOR
COVID-19
Luckily,
with
proper
precautions,
we
can
prevent
the
spread
of
COVID-19.
Coronaviruses
like
SARS-CoV-2
transmit
from
person-to-person
through
liquid
droplets
that
come
out
when
you
cough
or
sneeze.
Soap,
hand
sanitizer
and
other
disinfectants
kill
coronaviruses,
so
frequent
washing
of
hands
can
limit
its
spread.
Touching
your
face
with
contaminated
hands
can
intro-
~
IN
SEVERE
DISEASE:
\
Difficulty
waking
Confusion
/r:
Bluish
face
or
lips
COMMON
SYMPTOMS:
Fever:
83-99%
Loss
of
Appetite:
40-84%
Fatigue:
44-70%
Loss
of
smell:
15
to
30%
Coughing
up
blood
Persistent
chest
pain
Shortness
of
breath:
31-40%
P
Decreased
white blood
cells
duce
the
virus
to
your
mucus
membranes,
so
it
is
important
to
keep
hands
away
from
your
eyes,
nose
and
mouth.
We
can
wear
cloth
masks
to
cover
the
mouth
and
nose,
which
prevent
our
respiratory droplets
from
spreading
via
cough
or
sneeze.
Furthermore,
we
can
take
actions
like
social
distancing
to
reduce
the
likelihood
of
infecting
those
around
us,
which
can
decrease
the
spread
of
the
disease.
Cough:
59-82%
Coughing
up
sputum:
28-33%
Kidney
failure
Muscle
aches/pain:
11-35%
High
fever
Figure
2:
Symptoms
of
Coronavirus
Disease.
)
Testing
for
SARS-CoV-2
Symptoms
of
COVID-19
may
include
fever,
cough,
and
shortness
of
breath.
In
severe
cases,
patients
may
have
pneumonia,
respiratory
distress,
and/or
kidney
failure
(Figure
2).
Sadly,
this
infection
can
be
fatal.
Treatment
for
COVID-19
includes
rest,
fluids,
and
over-the-counter
cold
medications.
The
continued
development
of
vaccines
and
antiviral
medications
give
clinicians
the
necessary
tools
to
combat
infection.
If
you
are
exhibiting
symptoms
of
COVID-19,
seek
medical
attention
from
your
doctor
to
be
tested
for
the
virus.
There
are
two
types
of
diagnostic
tests
to
confirm
COVID-19
infection
-
Reverse
Transcription
PCR
(RT-PCR)
and
Enzyme-
Linked
Immunosorbent
Assay
(ELISA).
RT-PCR
tests
are
currently
in
use
by
public
health
[aboratories
around
the
world
for
the
presence
of
the
viral
genome,
signifying
active
infection.
Because
RT-PCR
is
extremely
sensitive
and
can
detect
minute
amounts
of
the
virus,
it
is
an
ideal
assay
to
detect
active
SARS-CoV-2
infections.
A
positive
test
does
not
mean
that
a
pa-
tient
will
become
seriously
ill;
however,
these
diagnoses
are
important
as
they
allow
epidemiologists
to
trace
and
limit
the
spread
of
COVID-19.
The
virus
responsible
for
COVID-19,
SARS-CoV-2,
does
not
integrate
itself
into
the
human
genome
during
infection
(like
HIV).
Once
the
patient’'s
immune
system
has
cleared
the
infection,
no
viral
nucleic
acid
remains
in
the
body
which
makes
the
RT-PCR
test
ineffective.
However,
the
antibodies
generated
to
fight
off
the
infection
remain
in
the
body
after
the
patient
has
healed.
The
ELISA
is
used
to
identify
the
presence
of
these
antibodies
in
patients,
signifying
that
a
person
had
been
previously
infected
by
the
virus.
However,
since
the
body
takes
several
days
to
produce
these
antibodies,
the
ELISA
cannot
detect
infected
people
before
clinical
symptoms
arise.
The
RT-PCR
Test
for
SARS-CoV-2
In
1984,
Dr.
Kary
Mullis
revolutionized
the
field
of
molecular
biology
when
he
devised
a
simple
and
elegant
method
to
copy
specific
pieces
of
DNA.
Mullis
recognized
that
he
could
replicate
DNA
in
vitro
using
short,
synthetic
DNA
oligonucleotides
(known
as
primers)
and
DNA
Polymerase
|
in
a
process
similar
to
DNA
replication
in
a
cell's
nucleus.
Furthermore,
because
researchers
can
customize
the
primers
to
target
a
specific
gene,
this
method
would
allow
for
the
rapid
amplification
of
a
selected
DNA
sequence.
For
the
development
of
this
technique,
known
today
as
the
Polymerase
Chain
Reaction
(or
PCR),
Mullis
was
awarded
the
Nobel
Prize
in
Chemistry
in
1993.
To
perform
PCR, purified
double-stranded
DNA
is
mixed
with
primers,
a
thermostable
DNA
polymerase
(Tag)
and
nucleo-
tides
(Figure
3).
Then,
the
mixture
is
heated
to
94°C
to
denature
the
DNA
duplex
(i.e.,
unzip
it
into
single
strands).
Next,
the
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all
rights
reserved.
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NUCLEIC
ACID
TESTING
FOR
COVID-19
I\
e
B
(14
A
PX]
Target
Sequence
—
5'wvams=————wm—
WV
3
t
_
Separation
of
3
W
M
5
two
DNA
strands
*
mm
=
Primer1
=
Primer2
S5'wwass——
A
3
Denature
¢
94°C
3
W
MW
5
-—
9
Y
=
O
5
wammm——
A
3
_l
¢
>
j
Anneal
.
5'™C
2
primers
L
|:3
>
_1
40°C
-
65°C
i
'
—)
5'wamms—
A
3
|
Extension
{“’
3'
5"
'
4_}
72°C
~N
5
—
S
M
A
3
()]
3'
e
5
e
5'
S
5
CE—)
5
T
3
O
3
M
mm—
AW
5
e
5
5'CE——
5
/W
—
A
3
3
e
5
<4mmmreeeem
5'
™M
5'
e
3
St
3'
@
3
O
5
€
S
@)
>
5
'
—)
5
e
3'
U
3
=———mm5'
3’
5’
5
(e
—
St
3'
3'
mwamms—
A
5
e
5
Figure
3:
DNA
Amplification
by
the
Polymerase
Chain
Reaction
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E
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EDVO-Kit
#123
NUCLEIC
ACID
TESTING
FOR
COVID-19
sample
is
then
cooled
to
45°C-60°C,
allowing
the
primers
to
base
pair
with
the
target
DNA
sequence
(called
“annealing”).
Lastly,
the
temperature
is
raised
to
72°C,
the
optimal
temperature
at
which
Tag
polymerase
will
extend
the
primer
to
syn-
thesize
a
new
strand
of
DNA.
Each
“PCR
cycle”
(denaturation,
annealing,
extension)
doubles
the
amount
of
the
target
DNA
in
less
than
five
minutes
(summarized
in
Figure
3).
In
order
to
produce
enough
DNA
for
analysis,
twenty
to
forty
cycles
may
be
required.
To
simplify
this
process,
a
specialized
machine,
called
a
“thermal
cycler”
or
a
“PCR
machine”,
was
created
to
rapidly
heat
and
cool
the
samples.
Because
of
its
ease
of
use
and
its
ability
to
rapidly
amplify
DNA,
PCR
has
become
indis-
pensable
in
medical
diagnostic
laboratories
for
the
detection
of
pathogens.
Unfortunately,
since
Tag
polymerase
is
a
DNA-dependent
DNA
polymerase,
it
cannot
use
the
RNA
genome
of
SARS-CoV2
as
a
template.
In
order
to
detect
COVID-19
using
PCR,
Reverse
Transcriptase
(RT)
is
used
to
synthesize
complementary
DNA
(cDNA)
copies
of
the
RNA
genome.
A
small
amount
of
the
cDNA
is
mixed
with
Taq
polymerase,
dNTPs
and
primers
for
amplification
by
PCR.
Because
RT-PCR
is
extremely
sensitive
and
can
detect
very
low
levels
of
the
virus,
it
is
considered
the
“gold
standard”
for
SARS-CoV-2
detection.
However,
since
RT-PCR
tests
are
performed
in
a
medical
diagnostic
laboratory,
it
may
take
several
days
to
get
the
results,
even
though
the
actual
test
takes
a
few
hours.
Commonly
used
RT-PCR
tests
combine
three
primer
sets
in
one
PCR
test,
which
is
known
as
multiplex
PCR.
The
first
two
sets
of
primers
target
regions
in
the
SARS-CoV-2
N
protein.
As
an
internal
control,
the
third
set
of
primers
amplify
the
hu-
man
housekeeping
gene
RNase
P
(RP).
When
combined
in
a
PCR sample,
the
three
sets
of
primers
produce
DNA
fragments
of
different
lengths
depending
upon
whether
the
SARS-CoV-2
virus
is
present
in
a
patient
sample.
In
order
to
analyze
this
sample,
scientists
may
use
a
technique
called
agarose
gel
electrophoresis
to
separate
DNA
frag-
ments
according
to
size.
In
electrophoresis,
mixtures
of
amplified
DNA
molecules
are
added
into
depressions
(or
“wells")
within
a
gel,
and
then
an
electrical
current
is
passed
through
the
gel.
Because
the
sugar-phosphate
backbone
of
DNA
has
a
strong
[/~
negative
charge,
the
current
drives
the
DNA
through
the
gel
A
(-)
8
(-)
towards
the
positive
electrode
(Figure
4A).
1
2
3
465
6
1
23
45
6
e
|
e
e
f
e
e
e
e
f
e
f
At
first
glance,
an
agarose
gel
appears
to
be
a
solid
at
room
temperature.
On
the
molecular
level,
the
gel
contains
small
=
channels
through
which
the
DNA
can
pass.
Small
DNA
fragments
-
move
through
these
holes
easily,
but
large
DNA
fragments
have
a
more
difficult
time
squeezing
through
the
tunnels.
Because
molecules
with
dissimilar
sizes
travel
at
different
speeds,
they
become
separated
and
form
discrete
“bands”
within
the
gel.
(+)
(+)
After
the
current
is
stopped,
the
bands
can
be
the
bands
can
be
Figure
4:
visualized
using
a
stain
that
sticks
to
DNA
(Figure
4B).
Overview
of
Agarose
Gel
Electrophoresis
In
this
simulated
medical
test,
we
will
use
electrophoresis
to
/
analyze
samples
from
three
patients
who
have
symptoms
of
COVID-19.
Samples
were
collected
using
a
nasopharyngeal
swab,
the
DNA
was
extracted,
and
then
analyzed
using
RT-PCR.
A
diagnosis
is
made
after
performing
electrophoresis
on
the
samples.
For
the
patients
that
have
been
infected
with
the
virus,
the
test
will
detect
both
the
viral
genome
and
the
internal
control,
resulting
in
three
bands
on
the
gel
(two
from
the
SARS-CoV
N
gene
and
one
from
the
human
control
gene).
In
contrast,
a
patient
who
was
not
infected
with
SARS-CoV-2
will
only
have one
band
on
the
gel
from
the
internal
control.
Figures:
Figure
1:
Picture
of
a
coronavirus:
https://en.wikipedia.org/wiki/Coronavirus#/media/File:3D_medical
animation_coronavirus_structure.jpg
/
CC
BY-SA
(https://cre-
ativecommons.org/licenses/by-sa/4.0)
Figure
2:
https.//en.wikipedia.org/wiki/Coronavirus_disease_2019#/media/File:Symptoms_of
coronavirus_disease_2019
_3.0.svg
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1989-2021
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reserved.
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NUCLEIC
ACID
TESTING
FOR
COVID-19
I\
e
B
(14
A
PX]
Experiment
Overview
EXPERIMENT
OBJECTIVE:
SARS-CoV-2
is
a
novel
coronavirus
that
has
caused
a
worldwide
outbreak
of
respiratory
disease
beginning
in
2019.
In
this
simulated
medical
test,
we
will
use
RT-PCR
to
detect
the
presence
of
the
SARS-CoV-2
virus
in
blood
sample
from
three
pa-
tients
with
symptoms
of
COVID-19
LABORATORY
SAFETY
1.
Gloves
and
goggles
should
be
worn
routinely
as
good
laboratory
practice.
2.
Exercise
extreme
caution
when
working
with
equipment
that
is
used
in
conjunction
with
the
heating
and/or
melting
of
reagents.
Wear
gloves
3.
DO
NOT
MOUTH
PIPET
REAGENTS
-
USE
PIPET
PUMPS.
and
safety
goggles
|
4.
Exercise
caution
when
using
any
electrical
equipment
in
the
laboratory.
b
Always
wash
hands
thoroughly
with
soap
and
water
after
handling
reagents
or
biological
materials
in
the
laboratory.
LABORATORY
NOTEBOOKS:
Scientists
document
everything
that
happens
during
an
experiment,
including
experimental
conditions,
thoughts
and
ob-
servations
while
conducting
the
experiment,
and,
of
course,
any
data
collected.
Today,
you'll
be
documenting
your
experi-
ment
in
a
laboratory
notebook
or
on
a
separate
worksheet.
Before
starting
the
Experiment:
»
Carefully
read
the
introduction
and
the
protocol.
Use
this
information
to
form
a
hypothesis
for
this
experiment.
*
Predict
the
results
of
your
experiment.
During
the
Experiment:
»
Record
your
observations.
After
the
Experiment:
»
Interpret
the
results
-
does
your
data
support
or
contradict
your
hypothesis?
«
If
you
repeated
this
experiment,
what
would
you
change?
Revise
your
hypothesis
to
reflect
this
change.
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E
K®
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part
of
this
document
is
permitted
for
non-profit
educational
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©
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Inc,,
all
rights
reserved.
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EDVO-Kit
#123
NUCLEIC
ACID
TESTING
FOR
COVID-19
Experiment
Overview
Prepare
1
agarose
gel
in
casting
tray.
MODULE
I:
Agarose
Gel
Electrophoresis
Time
required:
See
Table
C
]
2
Remove
end
caps
&
comb,
then
submerge
Quick
Reference
for
EDVO-Kit
#123
gel
under
buffer
in
electrophoresis
Size
of
gel
Groups
Placement
of
Wells
required
chamber.
casting
tray
per
gel
comb
per
group
7x7cm
1
group
1st
set
of
notches
6
10x7cm
|
1group
1st
set
of
notches
6
14x7cm
|
2groups
|
1stand
3rd
sets
of
notches
6
—
Load
each
3
sample
in
>
:
consecutive
wells
_
+
=
MODULE
II:
Staining
Agarose
4
Auachsafety
.
™
cover
&
connect
GEIS
USIng
FIaShBlue
|eads
o
power
source
-
to
conduct
electrophoresis.
Time
required:
30
min.
(
fter
electrophoresis
Related
EDVOTEK®
Instructional
Videos
transfer
gel
for
Stalnlng
EDVOTEK®
INSTRUCTIONAL
VIDEO:
EDVOTEK®
INSTRUCTIONAL
VIDEO:
PREPARING
AN
AGAROSE
Gel For
Electr
he,
sis
MEASURING
!
IQUIDS
/
With
An
Adjus
bu,
EDVOTEK®
INSTRUCTIONAL
VIDEO
Volume
Micre
r
,T.
STAINING
with
FlashBlue™
EDVOTEK®
INSTRUCTIONAL
VIDEO:
:
PERFORMING
'
Agarose
Gel
F/=Ztrg
"horesis
[
6
Analysis
on
.
—
white
light
g
"
www.youtube.com/edvotekinc
)
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1989-2021
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Inc.,,
all
rights
reserved.
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NUCLEIC
ACID
TESTING
FOR
COVID-19
10
Module
I:
Agarose
Gel
Electrophoresis
Concentrated
buffer
water
@
Z.O
1:00
—3
Caution!
Flask
will
be
HOT!
I\
e
B
(14
A
PX]
CASTING
THE
AGAROSE
GEL
1.
DILUTE
concentrated
50X
Electrophoresis
buffer
with
distilled
water
(refer
to
Table
A
for
correct
volumes
depending
on
the
size
of
your
gel
casting
tray).
2.
MIX
agarose
powder
with
buffer
solution
in
a
250
mL
flask
(refer
to
Table
A).
3.
DISSOLVE
agarose
powder
by
boiling
the
solution.
MICROWAVE
the
solution
on
high
for
1
minute.
Carefully
REMOVE
the
flask
from
the
microwave
and
MIX
by
swirling
the
flask.
Continue
to
HEAT
the
solution
in
15-second
bursts
until
the
agarose
is
completely
dis-
solved
(the
solution
should
be
clear
like
water).
4.
COOL
agarose
to
60
°C
with
careful
swirling
to
promote
even
dissipation
of
heat.
5.
While
agarose
is
cooling,
SEAL
the
ends
of
the
gel-casting
tray
with
the
rubber
end
caps.
PLACE
the
well
template
(comb)
in
the
appropriate
notch.
6.
POUR
the
cooled
agarose
solution
into
the
prepared
gel-casting
tray.
The
gel
should
thoroughly
solidify
within
20
minutes.
The
gel
will
stiffen
and
become
less
transparent
as
it
solidifies.
7.
REMOVE
end
caps
and
comb.
Take
particular
care
when
removing
the
comb
to
prevent
damage
to
the
wells.
EDVOTEK,
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©
Fax
202.370.1501
Wear
gloves
d
safet
I
and
safety
goggles
|
7
REMINDER:
This
experiment
requires
0.8%
agarose
gels
cast
with
6
wells.
J
Table
.
A
Individual
0.8%
UltraSpec-Agarose™
Gels
Size
of
Gel
|
Concentrated
Amt
of
TOTAL
Casting
tray
|
Buffer
(50x)
+
Water
+
Agarose
=
Volume
7X7cm
0.6
mL
0.24¢g
30
mL
10x7
cm*
09mL
036g
45
mL
14x7cm
1.2mL
048¢g
60
mL
*Recommended
gel
volume
for
the
EDGE™
Integrated
Electrophoresis
System.
(Cat.
#500).
info@edvotek.com
*
www.edvotek.com
Duplication
of
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part
of
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is
permitted
for
non-profit
educational
purposes
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©
1989-2021
EDVOTEK,
Inc,,
all
rights
reserved.
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EDVO-Kit
#123
NUCLEIC
ACID
TESTING
FOR
COVID-19
Module
I:
Agarose
Gel
Electrophoresis
8.
POUR
1X
Diluted
Buffer.
11.
RUNNING
THE
GEL
s
8.
PLACE
the
gel
(still
on
the
tray*)
into
the
electrophoresis
chamber.
COVER
the
gel
with
1X
REMINDER:
Electrophoresis
Buffer
(See
Table
B
for
recommended
volumes).
The
gel
should
be
com-
Bifaor;eplgsdrlr?agk?e
pletely
submerged.
sure
the
gel
is
9.
PUNCTURE
the
foil
overlay
of
the
QuickStrip™
with
a
pipet
tip.
LOAD
the
entire
sample
properly
oriented
(35
pL)
into
the
well
in
the
order
indicated
by
Table
1,
at
right.
in
the
apparatus
chamber.
10.
PLACE
safety
cover
on
the
unit.
CHECK
that
the
gel
is
properly
oriented.
Remember,
the
DNA
samples
will
migrate
toward
the
positive
(red)
electrode.
11.
CONNECT
leads
to
the
power
source
and
PERFORM
electrophoresis
(See
Table
1:
Gel
Loading
Table
C
for
time
and
voltage
guidelines).
Allow
the
tracking
dye
to
migrate
Lane
Tube
sample
at
least
3
cm
from
the
wells.
12.
After
electrophoresis
is
complete,
REMOVE
the
gel
and
casting
tray
from
1
TubeA
|
DNA
Standard
Marker
the
electrophoresis
chamber.
2
TubeB
|
Negative
Control
3
Tube
C
Positive
Control
PROCEED
to
Module
Il:
Staining
Agarose
Gels
Using
FlashBlue™.
4
TubeD
|
Patient
1
Sample
5
Tube
E
Patient
2
Sample
6
Tube
F
Patient
3
Sample
Table
Tale
Time
and
Voltage
Guidelines
B
1x
Electrophoresis
Buffer
(Chamber
Buffer)
C
(0.8%
Agarase
Gel)
Dilution
£lectrophoresis
Model
£DVOTEK
Total
Volume
50x
Conc.
Distilled
EDGE™
M12
&
M36
Model
#
Required
Buffer
+
Woater
Volts
Min/Max
(minutes)
Min/Max
(minutes)
EDGE™
150
mL
3mL
147
mL
150
10/20
20/35
M36
1000
mL
20
mL
980
mL
100
15/25
40/60
1.800.EDVOTEK
*
Fax
202.370.1501
-
info@edvotek.com
-
www.edvotek.com
Duplication
of
any
part
of
this
document
is
permitted
for
non-profit
educational
purposes
only.
Copyright
©
1989-2021
E
DVOT
E
K®
EDVOTEK,
Inc,,
all
rights
reserved.
123.200824
11
77
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NUCLEIC
ACID
TESTING
FOR
COVID-19
EDVO-Kit
#123
Module
II:
Staining
Agarose
Gels
Using
FlashBlue™
1.
DILUTE
10
mL
of
10x
concentrated
FlashBlue™
with
90
mL
of
water
in
a
flask
and
MIX
well.
2.
REMOVE
the
agarose
gel
and
casting
tray
from
the
electrophoresis
chamber.
SLIDE
the
gel
off
of
the
casting
tray
into
a
small,
clean
gel-staining
tray.
3.
COVER
the
gel
with
the
1x
FlashBlue™
stain
solution.
STAIN
the
gel
for
5
minutes.
For
best
results,
use
an
orbital
shaker
to
gently
agitate
the
gel
while
staining.
STAINING
THE
GEL
FOR
Wear
gloves
LONGER
THAN
5
MINUTES
WILL
REQUIRE
EXTRA
DESTAINING
TIME.
and
safety
goggles
4.
TRANSFER
the
gel
to
a
second
small
tray.
COVER
the
gel
with
water.
DESTAIN
for
at
least
20
minutes
with
gentle
shaking
(longer
periods
will
yield
better
results).
Frequent
changes
of
the
water
will
accelerate
de-
staining,.
5.
Carefully
REMOVE
the
gel
from
the
destaining
liquid.
VISUALIZE
results
using
a
white
light
visualization
system.
DNA
will
appear
as
dark
blue
bands
on
a
light
blue
background.
6.
ESTIMATE
the
base
pair
length
of
each
fragment
by
comparing
the
distance
each
fragment
traveled
from
the
well
to
the
bottom
of
the
gel
to
the
distance
that
each
Standard
DNA
Marker
fragment
migrated.
For
a
more
accurate
size
calcula-
tion
you
can
use
a
standard
curve
(see
Appendix
C).
7.
Fill
out
the
table
below.
Sample
Number
of
Fragments
|
Size
of
each
Fragment
7
6751,
3652, 2827,
1568,
1118,
825,
630
DNA
Standard
Marker
Negative
Control
Positive
Control
Patient
1
Patient
2
Patient
3
1.800.EDVOTEK
*
Fax
202.370.1501
-
info@edvotek.com
-
www.edvotek.com
E
DVOT
E
K®
Duplication
of
any
part
of
this
document
is
permitted
for
non-profit
educational
purposes
only.
Copyright
©
1989-2021
EDVOTEK,
Inc.,,
all
rights
reserved.
123.200824
12
78
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COVID
Testing
Lab
Questions
Include
a
picture
of
your
gel
along
with
the
answers
to
the
following
questions.
Some
of
the
bands
in
the
gel
may
be
faint,
but
you
should
be
able
to
see
enough
of
them
to
give
you
the
information
that
you
need
for
the
following
questions.
Increasing
the
brightness
or
contrast
of
pictures
while
looking
at
the
gels
may
help.
1.
Label
the
gel
with
the
following:
Lane/well
number.
The
tube
letter
that
is
in
each
well
and
what
each
sample
is.
The
positive
and
negative
ends
of
the
gel.
The
sizes
of
the
standard
marker
fragments.
The
known
sizes
are
6751,
3652,
2827,
1568,
1118,
825,
and
630
base
pairs.
You
may
not
be
able
to
see
all
of
the
bands,
so
refer
to
the
picture
below.
Indicate
which
size
corresponds
to
each
of
the
bands
in
your
marker
lane.
e.
The
approximate
sizes
of
the
bands
that
you
can
see
in
each
sample.
Do
this
by
comparing
the
bands
in
each
sample
to
the
standard
marker
fragments.
eooh
2.
Other
than
COVID-19,
what
are
some
other
illnesses
that
coronaviruses
are
known
to
cause?
3.
What
are
some
of
the
symptoms
of
COVID-19?
4.
The
human
genome
is
made
of
DNA.
What
is
the
genome
of
coronaviruses
made
of?
5.
In
coronaviruses,
what does
the
nucleocapsid
(N)
protein
do?
6.
What
does
RT-PCR
stand
for
and
what
is
this
test
designed
to
identify
when
testing
for
COVID-19?
79
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7.
Inthis
specific
RT-PCR
test,
how
many
genetic regions
are
targeted
(how
many
primer
sets
are
used)?
8.
Describe
each
of
the
genetic
regions
that
this
RT-PCR
test
targets.
9.
On
your
gel,
how
many
bands
do
you
expect
to
see
for
someone
that
does
not
have
COVID-19?
10.
On
your
gel,
how
many
bands
do
you
expect
to
see
for
someone
that
does
have
COVID-
197
11.
What
is
the
diagnosis
for
patient
1?
Explain.
12.
What
is
the
diagnosis
for
patient
2?
Explain.
13.
What
is
the
diagnosis
for
patient
3?
Explain.
Includes
EDVOTEK's
All-NEW
\
DNA
Standard
Marker
|
-
Better
separation
+
Easier
band
measurements
«
No
unused
bands
({1
(N
NEW
DNA
Standard
ladder
sizes:
Qfl,
3652,
2827,
1568, 1118,
825,
630
80
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Lab
#9
Genetics
in
the
News
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GOALS
1.
Observe
how
frequently
genetics
appears
in
the
popular
press.
2.
Observe
how
genetics
impacts
humanity.
3.
Practice
public
speaking.
We
are
constantly
being
exposed
to
news
articles
about
genetic
breakthroughs.
What
are
some
of
these
recent
advances
and
how
will
they
affect
our
lives?
EXERCISE:
Find
a
reputable
news
article
in
the
popular
press
(CNN,
BBC
News,
Time
Magazine,
New
York
Times,
TheScientist,
etc.)
that
summarizes
a
recently
published
scientific
paper.
(Recent
means
within
the
last
couple
of
months
to
a
year).
The
topic
needs
to
be
related
to
genetics.
Step
2:
Have
the
articl
T
instruct
Submit
your
article
to
Canvas
and
have
your
instructor
approve
your
choice.
Submit
the
link
of
the
popular
press
article.
|
will
be
looking
to
see
that
the
articles
are
focused
on
genetics
and
checking
the
topic
against
what
other
students
have
submitted.
Only
one
person
in
each
lab
class
may
present
a
topic,
so
first
come,
first
served.
:
Summarize
the
Article
in
1-2
paraaraphs.
EXERCISE:
Read
the
news
article
and
write
a
summary
of
it
in
your
own
words.
This
should
be
about
1-2
paragraphs
long,
single
spaced.
AVOID
PLAGIARISM.
Your
summary
should
address
the
following
points:
What
was
the
important
question
being
asked?
What
did
we
know
before
this
study?
What
did
the
researchers
do?
What
did
the
researchers
learn?
How
will
this
impact
society?
Step
4:
P
I
findina
to
the
cl
EXERCISE:
Prepare
a
3-5
minute
oral
summary
of
your
readings
and
deliver
this
to
your
lab
mates.
You
do
not
need
to
prepare
a
PowerPoint
presentation.
You
will
do
a
zoom
presentation
for
the
class
and
tell
us
the
answers
to
the
questions
above.
You
may
have
notes
with
you
during
your
presentation
but
please
do
not
read
a
script.
Step
5:
Ask
vour
classmates
questions,
EXERCISE:
Everyone
in
each
lab
will
give
a
presentation
on
the
same
day.
You
are
expected
to
actively
listen
to
your
classmates
and
ask
them
questions
about
the
articles
they
are
presenting.
|
expect
you
to
ask
at
least
one
question
to
a
classmate.
If
nobody
asks
a
student
a
qguestion,
|
will!
82
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