Question 10. After liquid atomization (nebulization) of DNA, it is necessary to repair the ends of the DNA molecules. This repair involves: Using T4 DNA polymerase to fill in the 3'-protruding ends. b. Digesting DNA fragments with a restriction enzyme so that all of the "sticky" (cohesive) ends are the same. Removing phosphate from the protruding 3' and 5' ends. Using T4 DNA polymerase to fill in the 3' recessed ends and digest the single-stranded 3' protruding ends. c. d.

Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
Section: Chapter Questions
Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...
icon
Related questions
icon
Concept explainers
Question
100%

Seeking to confirm the answer based on the information attached

We will "repair" ends of DNA fragments in this laboratory period and prepare them for cloning.
DNA molecules sheared by the liquid atomization process (nebulization) have different 5' and 3' ends.
About 80% of ends are protruding ends or blunt-ended at one end of DNA fragments. These ends cannot be
cloned into the cloning vector. To clone DNA fragments into a cloning vector, both ends of the DNA must
be blunt-ended and phosphorylated at 5' ends. Blunt phosphorylated ends are necessary for enzyme ligase
to ligate fragments into a plasmid vector.
4
Two enzymes will be used T₂ DNA polymerase and T, DNA kinase. Both enzymes are proteins coded by the
T₁ bacteriophage of E. coli. The enzymes were modified to be more useful for DNA cloning.
T4 DNA polymerase has two activities DNA synthesis and 3' exonuclease activity. DNA polymerase extends
3' recessed ends using the template 5' protruding ends. 3' exonuclease removes single-stranded
3' protruding ends. Both activities result in creating blunt-ended DNA fragments.
T4 polymerase was modified to have maximum activity at room temperature.
T₁ DNA kinase adds phosphate to 5' ends of DNA using ATP as the phosphate source. T4 kinase was
modified to have maximum activity at room temperature and prefer the blunt ends of DNA to add
phosphate.
Both enzymes can be used together in the same reaction.
Transcribed Image Text:We will "repair" ends of DNA fragments in this laboratory period and prepare them for cloning. DNA molecules sheared by the liquid atomization process (nebulization) have different 5' and 3' ends. About 80% of ends are protruding ends or blunt-ended at one end of DNA fragments. These ends cannot be cloned into the cloning vector. To clone DNA fragments into a cloning vector, both ends of the DNA must be blunt-ended and phosphorylated at 5' ends. Blunt phosphorylated ends are necessary for enzyme ligase to ligate fragments into a plasmid vector. 4 Two enzymes will be used T₂ DNA polymerase and T, DNA kinase. Both enzymes are proteins coded by the T₁ bacteriophage of E. coli. The enzymes were modified to be more useful for DNA cloning. T4 DNA polymerase has two activities DNA synthesis and 3' exonuclease activity. DNA polymerase extends 3' recessed ends using the template 5' protruding ends. 3' exonuclease removes single-stranded 3' protruding ends. Both activities result in creating blunt-ended DNA fragments. T4 polymerase was modified to have maximum activity at room temperature. T₁ DNA kinase adds phosphate to 5' ends of DNA using ATP as the phosphate source. T4 kinase was modified to have maximum activity at room temperature and prefer the blunt ends of DNA to add phosphate. Both enzymes can be used together in the same reaction.
Question 10. After liquid atomization (nebulization) of DNA, it is necessary to repair the
ends of the DNA molecules. This repair involves:
C
a Using T4 DNA polymerase to fill in the 3'-protruding ends.
b. Digesting DNA fragments with a restriction enzyme so that all of the "sticky"
(cohesive) ends are the same.
c.
Removing phosphate from the protruding 3' and 5' ends.
d. Using T4 DNA polymerase to fill in the 3' recessed ends and digest the
single-stranded 3' protruding ends.
Transcribed Image Text:Question 10. After liquid atomization (nebulization) of DNA, it is necessary to repair the ends of the DNA molecules. This repair involves: C a Using T4 DNA polymerase to fill in the 3'-protruding ends. b. Digesting DNA fragments with a restriction enzyme so that all of the "sticky" (cohesive) ends are the same. c. Removing phosphate from the protruding 3' and 5' ends. d. Using T4 DNA polymerase to fill in the 3' recessed ends and digest the single-stranded 3' protruding ends.
Expert Solution
steps

Step by step

Solved in 4 steps

Blurred answer
Knowledge Booster
Molecular techniques
Learn more about
Need a deep-dive on the concept behind this application? Look no further. Learn more about this topic, biology and related others by exploring similar questions and additional content below.
Recommended textbooks for you
Human Anatomy & Physiology (11th Edition)
Human Anatomy & Physiology (11th Edition)
Biology
ISBN:
9780134580999
Author:
Elaine N. Marieb, Katja N. Hoehn
Publisher:
PEARSON
Biology 2e
Biology 2e
Biology
ISBN:
9781947172517
Author:
Matthew Douglas, Jung Choi, Mary Ann Clark
Publisher:
OpenStax
Anatomy & Physiology
Anatomy & Physiology
Biology
ISBN:
9781259398629
Author:
McKinley, Michael P., O'loughlin, Valerie Dean, Bidle, Theresa Stouter
Publisher:
Mcgraw Hill Education,
Molecular Biology of the Cell (Sixth Edition)
Molecular Biology of the Cell (Sixth Edition)
Biology
ISBN:
9780815344322
Author:
Bruce Alberts, Alexander D. Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter
Publisher:
W. W. Norton & Company
Laboratory Manual For Human Anatomy & Physiology
Laboratory Manual For Human Anatomy & Physiology
Biology
ISBN:
9781260159363
Author:
Martin, Terry R., Prentice-craver, Cynthia
Publisher:
McGraw-Hill Publishing Co.
Inquiry Into Life (16th Edition)
Inquiry Into Life (16th Edition)
Biology
ISBN:
9781260231700
Author:
Sylvia S. Mader, Michael Windelspecht
Publisher:
McGraw Hill Education