Sanger sequencing Flashcards
What is the sanger method
It is alternatively called chain terminator method and dideoxy method. It uses the E.coli enzyme DNA polymerase I to synthesize complementary copies of single-stranded DNA being sequenced
Explain what the sanger method involves in general
Using the template strand for replication, DNA polymerase I assembles the four deoxynucleoside triphosphates (dATP,dTTP,dCTP,dGTP- dNTPs) into a complementary polynucleotide strand that elongates in the 5’ to 3’ direction. To initiate DNA synthesis, DNA polymerase requires a primer annealed to the template. The DNA templates are obtained in sufficient quantity to sequence by cloning them in M13 vectors or by PCR- both yields single-stranded DNA.
What occurs in the sanger method if the DNA being sequenced is a restriction fragment
The primer can be a short DNA segment containing this restriction site annealed to the strand being replicated - the restriction fragment begins and ends with a restriction site.
Explain what the Klenow fragment is
DNA polymerase I’s 5’-3’ exonuclease activity is catalyzed by a separate active site from those which mediate its polymerase and 3’-5’ exonuclease activity. This is demonstrated by the observation that on proteolytic cleavage of the enzyme into 2 fragments, the larger C-terminal fragment which is known as the klenow fragment has the polymerase and 3’-5’ exonuclease activity, while the smaller N-terminal fragment has the 5’-3’ exonuclease activity. In the DNA sequencing only the klenow fragment is used to ensure that all the replicated chains have the same 5’ terminus
Explain the sanger method in detail
The DNA that is to be sequenced is incubated with the Klenow fragment of DNA polymerase I, a suitable primer, and the 4 dNTPs. Either at least one dNTP(usually dATP) or the primer is alpha-32P-labeled. Also, a small amount of the 2’,3’- dideoxynucleoside triphosphate (ddNTP) of one of the bases is added to the mixture.
What is the purpose of adding the ddNTP
When the ddNTP is added to the growing nucleotide chain in place of a nucleotide, the chain is terminated because of the absence of a 3’-OH group on the ddNTP.
Describe the automation of the sanger method
The sanger method has been automated to sequence large tracts of DNA, such as entire chromosomes- this accelerates the process. Fluorescence labeling techniques are used. The most widely used fluorescence labeling technique involves each of the four ddNTPs used to terminate chain extension is covalently linked to a fluorescing dye. The chain-extension reactions are carried out in a single vessel containing all 4 ddNTPs labeled with fluorescent dye and the resulting fragment mixture is exposed to gel electrophoresis in a single lane. As each fragment exists the gel its terminal base is identified according to its characteristic fluorescence spectrum by a laser-activated fluorescence detection system.
