Lecture 29: Chain termination, DNA sequencing
1. Explain the role played by electrophoresis in determining DNA
sequences.
2. What type of reagent is used to achieve selective chain termination
in DNA sequencing studies?
3. DNA sequencing is commonly done with single-stranded DNA as
the starting material. Do you think this is strictly necessary.
Explain the reasoning behind your answer.
4. Why is it important to use a polymerase that lacks 5' to 3'
exonuclease activity (such as the Klenow fragment of DNA polymerase
I) when doing DNA sequencing?
5. You have a cDNA that is about 600 nucleotides long cloned in
M13 virus that has been engineered to include a polylinker site.
Describe the primers that you would need to determine its entire
sequence.
6. Why is it necessary to use four parallel electrophoretic lanes
when determining DNA sequence by "standard" methods?
What newer methods have eliminated this requirement?
7. Explain how dideoxyribonucleotide triphosphates terminate chain
growth.
8. What prevents all growing chains from being terminated at the
same length?
9. What is an open reading frame and why is its identification
important during DNA sequencing?
10. Sanger's early analysis of DNA sequence in the single stranded
DNA bacteriophage phi-X174 revealed overlapping open reading frames.
What is the significance of this discovery?
11. You are sequencing the cDNA that codes for a particular enzyme
in mice. As a check on the accuracy of your procedures, you do
sequence analysis on two separate clones both prepared from the
mRNA of the same mouse. The two sequences correspond exactly,
except at one nucleotide about halfway through the coding sequence,
where you obtain a C in one of the samples and a T in the other.
b. You transfer both clones to an expression vector, and they
both produce equally functional enzyme. What is the most likely
explanation for how this can happen?
c. Would you expect to see any differences in the amino acid sequences
of the two enzymes? Explain your answer.
d. In part b, one of the expression vectors fails to produce a
functional enzyme. How would this affect your answers to parts b and c?
e. You have a pair of unique sequence oligonucleotide primers
that allow you to use PCR to amplify a sequence 150 nucleotides
long that contains the C versus T site, using genomic DNA from the same
mouse as your starting material. You then do sequencing on the PCR product,
using the primer that hybridizes to the antisense strand as your
sequencing primer. What do you expect to see on your sequencing
gel at the C versus T site? (Note that this question requires
a knowledge of PCR from Lecture 30).
12. You are sequencing using the sense strand of a cDNA that has
been cloned into an M13 viral vector as the template. How does
the data that you read from your sequencing gel need to be modified
to obtain the correct sequence for the sense strand of the cDNA?
What term is used to describe the sequence that you have read
from your gel?
13. Why does the sequence you read from the gel in Sanger dideoxy
sequencing not begin with the sequence of the primer?
14. What are the advantages of using fluorescent labels rather
than radioactive labels for sequencing?
15. When sequencing with a primer for a vector with a multi-cloning
site, you encounter the sequence GAATTC ahead of the cloned sequence
that you are expecting. Explain how this might happen.
16. The RNA coding sequence for the tryptophan operon attenuator
peptide (lecture 35) is
b. Same as a, except using the sense strand of the DNA as the
template.
c. Same as a, except containing a frameshift mutation that causes
the formation of a stop codon (UGA) near the middle of the sequence.
The stop codon is to be formed by frame-shifting, and not by the
direct insertion or deletion of a base at the site of the stop
codon.
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a. How would you interpret the data?
a. Sketch a sequencing gel showing the sequence that would be
obtained if you used the antisense strand of the DNA as the template
and a primer that hybridized to the DNA just beyond the end of
the coding sequence.
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