As you learned in this chapter,restriction enzymes are sophisticated“scissors” that geneticistsand molecular biologists routinelyuse to cut DNA for recombinant DNAexperiments. A wide variety of onlinetools assist scientists working with restrictionenzymes and manipulating recombinantDNA for different applications. Herewe explore Webcutter and Primer3, twosites that make recombinant DNA experimentsmuch easier.Exercise I – Creating a RestrictionMap in WebcutterSuppose you had cloned and sequenceda gene and you wanted to design a probeapproximately 600 bp long that could beused to analyze expression of this gene indifferent human tissues by Northern blotanalysis. Internet sites such as Webcuttermake it relatively easy to design experimentsfor manipulating recombinant DNA.In this exercise, you will use Webcutter tocreate a restriction map of human DNAwith the enzymes EcoRI, BamHI, and PstI.1. Access Webcutter at http://www.firstmarket.com/cutter/cut2.html.Go to the Study Area for Essentialsof Genetics, and open the ExploringGenomics exercise for this chapter.Copy the sequence of cloned humanDNA found there, and paste it into thetext box in Webcutter.2. Scroll down to “Please indicate whichenzymes to include in the analysis.”Click the button indicating, “Use onlythe following enzymes.” Select therestriction enzymes EcoRI, BamHI, andPstI from the list provided, and thenclick “Analyze sequence.” (Note: Usethe command, control, or shift key toselect multiple restriction enzymes.)3. After examining the results provided byWebcutter, create a table showing thenumber of cutting sites for each enzymeand the fragment sizes that wouldbe generated by digesting with eachenzyme. Draw a restriction map indicatingcutting sites for each enzyme withdistances between each site and thetotal size of this piece of human DNA.Exercise II – Designing aRecombinant DNA ExperimentNow that you have created a restrictionmap of your piece of human DNA, youneed to ligate the DNA into a plasmidDNA vector that you can use to make yourprobe (molecular biologists often referto this as subcloning). To do this, youwill need to determine which restrictionenzymes would best be suited for cuttingboth the plasmid and the human DNA.1. Referring back to the Study Area andthe Exploring Genomics exercise forthis chapter, copy the plasmid DNAsequence from Exercise I into the textbox in Webcutter and identify cuttingsites for the same enzymes you used inExercise I. Then answer the followingquestions Question: Draw a simple diagram showing thecloned DNA inserted into the plasmid,and indicate the restrictionenzymecutting site(s) used to createthis recombinant plasmid.
Molecular Techniques
Molecular techniques are methods employed in molecular biology, genetics, biochemistry, and biophysics to manipulate and analyze nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), protein, and lipids. Techniques in molecular biology are employed to investigate the molecular basis for biological activity. These techniques are used to analyze cellular properties, structures, and chemical reactions, with a focus on how certain molecules regulate cellular reactions and growth.
DNA Fingerprinting and Gel Electrophoresis
The genetic makeup of living organisms is shown by a technique known as DNA fingerprinting. The difference is the satellite region of DNA is shown by this process. Alex Jeffreys has invented the process of DNA fingerprinting in 1985. Any biological samples such as blood, hair, saliva, semen can be used for DNA fingerprinting. DNA fingerprinting is also known as DNA profiling or molecular fingerprinting.
Molecular Markers
A known DNA sequence or gene sequence is present on a chromosome, and it is associated with a specific trait or character. It is mainly used as a genetic marker of the molecular marker. The first genetic map was done in a fruit fly, using genes as the first marker. In two categories, molecular markers are classified, classical marker and a DNA marker. A molecular marker is also known as a genetic marker.
DNA Sequencing
The most important feature of DNA (deoxyribonucleic acid) molecules are nucleotide sequences and the identification of genes and their activities. This the reason why scientists have been working to determine the sequences of pieces of DNA covered under the genomic field. The primary objective of the Human Genome Project was to determine the nucleotide sequence of the entire human nuclear genome. DNA sequencing selectively eliminates the introns leading to only exome sequencing that allows proteins coding.
As you learned in this chapter,
restriction enzymes are sophisticated
“scissors” that geneticists
and molecular biologists routinely
use to cut DNA for recombinant DNA
experiments. A wide variety of online
tools assist scientists working with restriction
enzymes and manipulating recombinant
DNA for different applications. Here
we explore Webcutter and Primer3, two
sites that make recombinant DNA experiments
much easier.
Exercise I – Creating a Restriction
Map in Webcutter
Suppose you had cloned and sequenced
a gene and you wanted to design a probe
approximately 600 bp long that could be
used to analyze expression of this gene in
different human tissues by Northern blot
analysis. Internet sites such as Webcutter
make it relatively easy to design experiments
for manipulating recombinant DNA.
In this exercise, you will use Webcutter to
create a restriction map of human DNA
with the enzymes EcoRI, BamHI, and PstI.
1. Access Webcutter at http://www
.firstmarket.com/cutter/cut2.html.
Go to the Study Area for Essentials
of Genetics, and open the Exploring
Genomics exercise for this chapter.
Copy the sequence of cloned human
DNA found there, and paste it into the
text box in Webcutter.
2. Scroll down to “Please indicate which
enzymes to include in the analysis.”
Click the button indicating, “Use only
the following enzymes.” Select the
restriction enzymes EcoRI, BamHI, and
PstI from the list provided, and then
click “Analyze sequence.” (Note: Use
the command, control, or shift key to
select multiple restriction enzymes.)
3. After examining the results provided by
Webcutter, create a table showing the
number of cutting sites for each enzyme
and the fragment sizes that would
be generated by digesting with each
enzyme. Draw a restriction map indicating
cutting sites for each enzyme with
distances between each site and the
total size of this piece of human DNA.
Exercise II – Designing a
Recombinant DNA Experiment
Now that you have created a restriction
map of your piece of human DNA, you
need to ligate the DNA into a plasmid
DNA vector that you can use to make your
probe (molecular biologists often refer
to this as subcloning). To do this, you
will need to determine which restriction
enzymes would best be suited for cutting
both the plasmid and the human DNA.
1. Referring back to the Study Area and
the Exploring Genomics exercise for
this chapter, copy the plasmid DNA
sequence from Exercise I into the text
box in Webcutter and identify cutting
sites for the same enzymes you used in
Exercise I. Then answer the following
questions
Question: Draw a simple diagram showing the
cloned DNA inserted into the plasmid,
and indicate the restrictionenzyme
cutting site(s) used to create
this recombinant plasmid.
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