DNA Flashcards
What did sanger use to sequence DNA ?
Klenow fragment of E.coli DNA polymerase 1.
why did sanger use this fragment ?
lacks 5 to 3 prime exonuclease activity
what substrate does the sanger sequencing method us e?
2’,3’ - dideoxynucleoside triphosphates (ddNTPs)
what is an oligonucleotide ?
a primer is a type of oligo..
what does DNA polymerase 1 do in the sanger sequencing method ?
catalyses the elongation of the primer, which is complementary to the 3’ end of the single stranded DNA to be sequenced..
why is alpha-P32- labeled dNTP used in the sanger sequencing method ?
its radioactivity allows the newly synthesised DNA to be visualised by autoradiography
what is added to each of the four tubes ?
alpha 32 phosphate labeled dNTP, an excess of the nonradioactive four dNTPS and a small amount of one of the ddNTPs
what does the length of each fragment correspond to ?
the distance from the 5’ -end of the primer to one of the adenine residues in the sequence (ddATP was added)..
how can the DNA sequence then be read?
after electrophoresis, the sequence can be read from an autoradiograph of the gel
what enzyme has 5’ to 3’ exonuclease activity and 3’ to 5’ proofreading activity ?
DNA polymerase 1, removes RNA primers and replacing them with DNA using its 5’ to 3’ polymerase activity ?
what activity does DNA polymerase 3 exhibit ?
5’ to 3’ polymerase activity and 3’ to 5’ proofreading activity
what does DNA ligase ?
joins adjacent okazaki fragments by forming the phosphodiester linkage between 3’ of one fragment and 5’ end of the subsequent fragment
what does DNA polymerase 1 do ?
joins together okazaki fragments after removing the RNA primer at the begging of each fragment, and replacing it with DNA
what is the substrate for elongation of DNA by DNA polymerase 3? what is the reaction called ?
deoxyribonucleoside 5’-triphosphate (dNTP), nucleotide group transfer reaction
what does DNA polymerase 2 do ?
DNA repair
