DNA sequencing Flashcards
Discuss the history of the development of DNA sequencing
Frederick Sanger determined the amino acid sequence of insulin, proving for the first time that proteins had definite sequences and this earned him the Nobel prize in Chemistry in 1958. Then he looked at sequencing RNA and then sequencing DNA. His methods for sequencing DNA earned him his second Nobel Prize in Chemistry in 1980.
There were many challenges that he had to face in the early 1970s when sequencing DNA: difficult to prepare pure samples of even ssDNA, REs were a new discovery and were not really understood and, bacteriophage thetaX-174 (providing 5386 bp of purifiable, ssDNA) was a natural source of material for Sanger’s early work yielding the 1st whole genome sequence. The problem with is genome is that it was too long, so the sequence assembly challenge was how to sequence large fragments and put them back together. This is when Sanger’s method came in.
What are the requirements for Sanger’s method
DNA polymerase is a replication enzyme that synthesizes a new strand that is complementary to the template ssDNA. It requires a primer that provides a free 3’-OH group, deoxynucleoside triphosphates (dNTPs) which are the nucleotides that are used to form the newly synthesized strand, and ddNTPs (dideoxynucleoside triphosphates) which are dNTPs that lack a free 3’-OH group. Sanger sequencing forms the basis of PCR.
Explain the Sanger sequencing method (Chain termination method)
dsDNA to make a ssDNA template. DNA polymerase links the 5’-hydroxyl group of the newly added nucleotide to the 3’-position of the end of the primer. Hydrolysis of NTPs provides energy. Sanger’s idea was to include in the mixture of dNTPs a fraction of molecules containing no free 3’-OH called ddNTPs. One reaction mixture contains dATP. dTTP, dGTP, dCTP and ddCTP. As primer extension proceeds, when the next unpaired base in the template is a guanine, the enzyme will randomly incorporate either dCTP, after which normal extension will continue or ddCTP, after which no more extension will occur(The other 3 reactions run parallel and either contain ddATP,ddTTP or ddGTP). This reaction produces a mixture of fragments of different lengths, each of which ends in ddCTP: the crucial point is that the fragments are nested. To determine the positions of the cytosine residues in the newly synthesized strand, it is sufficient to determine the lengths of the fragments. Polyacrylamide gel electrophoresis can separate oligonucleotides according to their lengths accurately enough for sequence determination.
In Sanger’s original procedure, the ddCTP carried a radioactive label and the gel was developed by autoradiography. To determine the positions of the three other nucleotides, it was necessary to run all 4 reactions, in parallel, each containing a ddNTP with a radioactive label. The four separate reactions are necessary because each nucleotide gives the same signal- the darkening of the film from the radioactive spot. Running the products of the four reactions in parallel lanes on the same gel allows the sequence to be read from the autoradiograph.
Explain the Maxam-Gilbert chemical cleavage method
This method for DNA sequencing, also developed in the 1970s, also worked by comparing nested fragments. A sample of ssDNA, labelled at its 5’-end, is cleaved by base-specific reagents. As in the Sanger’s method, polyacrylamide gel electrophoresis separates the fragments by size. The separate cleavage reactions produce fragments that share the 5’-labelled end but differ in the bases at which the specific 3’-cleavage occurs. The sequence can be read from an autoradiograph. The disadvantages of the Maxam-Gilbert chemical cleavage method are: no primed DNA- its applicability is inherently limited to sequences
adjacent to restriction sites or other fixed termini and it uses toxic reagents, notably of hydrazine, a neurotoxin.
Discuss the automation of DNA sequencing
It uses fluorescent dyes instead of radioactive labels as reporters- this was an important technical advance in sequencing (radioisotopes present health hazards in both use and disposal and are expensive). By attaching 4 different fluorescent dyes to the 4 ddNTPs, each fragment produced gives a different signal depending witch ddNTP terminated the extension, therefore all 4 reactions can be done in a single reaction and electrophoresis can separate the fragment in a single lane. A laser focused at a fixed point identifies the fragments as they pass. The result can be displayed as a four-colour chromatogram in which successive peaks correspond to successive bases in the sequence.
Explain how the automation of DNA sequencing measures quality
The phred score q measures sequencing accuracy: q=20 implies a probability of < = 1% error per base. A common unit of sequencing cost is US$ per 1000 bases determined to q=20 accuracy- a sequence of 1000 bases determined at an accuracy of q=20 would be expected to contain no more than 10 errors.
The phred score of a sequence determination is a measure of sequence quality. It specifies the probability that the base reported is correct. If p= the probability that a base is in error, then the corresponding phred score q= -10log(p). e.g. if q is 10 then p is 0.1 which means that 1 base in 10 is wrong. if q is 20 then p is 0.01 which means that 1 base in 100 is wrong.
