BIOC0005 - DNA SEQUENCING; TRANSGENE
EXPRESSION; PTMS; SYNTHETIC BIOLOGY EXAM
QUESTIONS AND ANSWERS WITH COMPLETE
SOLUTIONS VERIFIED LATEST UPDATE
What do the following reveal to us about DNA?
Chargaff's rule
Watson and Crick's structure
Nirenberg's genetic code
1:1 stoichiometric ratio of pyrimidine (C, T) and purine (A, G) bases.
Double-stranded structure of DNA and base-pairing rules: T-A and C-G
Triplet code of DNA bases and linear relationship with protein sequence
What are the fundamentals of the 'dideoxy chain termination' and 'chemical
modification and DNA cleavage' methods of DNA sequencing?
Define a fixed DNA start point.
Generate a set of radiolabelled DNA strands that start at that point and finish at every
possible base.
,Physically separate the nested set using a size-fractionation that can resolve strands
that differ by a single base.
Explain how Sanger sequencing works.
Method involves second-strand synthesis using a DNA polymerase acting on a specific
piece of single-stranded DNA.
An oligonucleotide primer is fixed at the 5' end and is the same used for every
synthesis.
Klenow fragment and all 4 dNTPs added. If one of the dNTPs is radioactive (such as
32P, 35S), the newly synthesized strands will be radioactive, and therefore detectable.
To create a nested set of chains, the reaction can be spiked using a ddNTP. A ratio of
20:1 dCTP:ddCTP (for example) will not block strand synthesis but results in occasional
chain terminations.
Carrying out four separate reactions (with each ddNTP) will generate all possible chain
terminations.
Size fractionation of the different radiolabelled strands on a denaturing polyacrylamide
gel. This has the resolution to separate DNA strands that differ by only one base. Gel is
then dried and exposed to X-ray film and film read from bottom to top.
What are some of the issues with Sanger's original DNA sequencing method?
, Involved radioactivity. Hazardous and limited 'shelf-life' (half-life of 32P is two weeks;
35S is three months).
'Klenow' DNA polymerase not a great enzyme (poor processivity, struggled with
secondary structures in template).
Labelling of a normal dNTP means that stalled chains also detected, in addition to
dideoxy terminations.
Required four separate reactions.
Pouring, loading, running, handling and drying of gels were all time-consuming and
technically difficult.
Dried gels required ~24 h exposure to X-ray film.
Maximum 'read' from a single film was ~300 bases.
Sequences had to be manually read and typed into a computer for analysis.
What are the improvements to Sanger's method?
Labelling using four fluorescent dyes instead of radioactivity. As dyes have different
emission maxima, can label ddNTPs directly.
EXPRESSION; PTMS; SYNTHETIC BIOLOGY EXAM
QUESTIONS AND ANSWERS WITH COMPLETE
SOLUTIONS VERIFIED LATEST UPDATE
What do the following reveal to us about DNA?
Chargaff's rule
Watson and Crick's structure
Nirenberg's genetic code
1:1 stoichiometric ratio of pyrimidine (C, T) and purine (A, G) bases.
Double-stranded structure of DNA and base-pairing rules: T-A and C-G
Triplet code of DNA bases and linear relationship with protein sequence
What are the fundamentals of the 'dideoxy chain termination' and 'chemical
modification and DNA cleavage' methods of DNA sequencing?
Define a fixed DNA start point.
Generate a set of radiolabelled DNA strands that start at that point and finish at every
possible base.
,Physically separate the nested set using a size-fractionation that can resolve strands
that differ by a single base.
Explain how Sanger sequencing works.
Method involves second-strand synthesis using a DNA polymerase acting on a specific
piece of single-stranded DNA.
An oligonucleotide primer is fixed at the 5' end and is the same used for every
synthesis.
Klenow fragment and all 4 dNTPs added. If one of the dNTPs is radioactive (such as
32P, 35S), the newly synthesized strands will be radioactive, and therefore detectable.
To create a nested set of chains, the reaction can be spiked using a ddNTP. A ratio of
20:1 dCTP:ddCTP (for example) will not block strand synthesis but results in occasional
chain terminations.
Carrying out four separate reactions (with each ddNTP) will generate all possible chain
terminations.
Size fractionation of the different radiolabelled strands on a denaturing polyacrylamide
gel. This has the resolution to separate DNA strands that differ by only one base. Gel is
then dried and exposed to X-ray film and film read from bottom to top.
What are some of the issues with Sanger's original DNA sequencing method?
, Involved radioactivity. Hazardous and limited 'shelf-life' (half-life of 32P is two weeks;
35S is three months).
'Klenow' DNA polymerase not a great enzyme (poor processivity, struggled with
secondary structures in template).
Labelling of a normal dNTP means that stalled chains also detected, in addition to
dideoxy terminations.
Required four separate reactions.
Pouring, loading, running, handling and drying of gels were all time-consuming and
technically difficult.
Dried gels required ~24 h exposure to X-ray film.
Maximum 'read' from a single film was ~300 bases.
Sequences had to be manually read and typed into a computer for analysis.
What are the improvements to Sanger's method?
Labelling using four fluorescent dyes instead of radioactivity. As dyes have different
emission maxima, can label ddNTPs directly.
