DNA sequencing early Flashcards
who came up with the first method for DNA sequencing and what was it called
sanger, chain termination
what does chain termination sequencing rely on
isolation of recomb. DNA clone that carries an insert
step 1 of chain termination sequencing
DNA has to be denatured (by heat +100C or ph NaOH change)
step 2 chain termination sequencing
sequencing primer that is complementary to the coloning vector
what is another name for a sequencing primer and what does it do
oligonucleotide is a single strand DNA that provides a 3’ OH end for the new DNA sequence
step 3 chain termination sequencing
a mixture of 4 dNTP’s are added to all of the tubes
step 4 chain termination sequencing
DNA polymerase is added to all four of the tubes * at least one of these is radioactively labeled
what is a chain determining neuclotide called and what are the diff. types used in chain termination sequencing
(ddNTP) dideoxynucleotide triphosphate , 4 types: ddCPT, ddATP, ddTTP, ddGTP
what is the difference between dNTP, and ddNTP
ddNTP, is missing the 3’OH group this prevents any addition of nucleotide after the ddNTP has been added
step 5 chain termination sequencing
adding one of the ddNTP’s to each of the tubes
when a ddCTP is used what is the last nucleotide before termination (is the same for all types of ddNTP’s)
the C would be the last nucleotide
what is the type of gel used for gel eletrolysis for chain termination sequencing
on a polyacrylamide gel - the resolution is very accurate
how would you read a chain termination sequencing gel
from bottom to top
what are the 3 limitations to chain termination sequencing
1) it was labor intensive to set up gels and manually read them 2)it could take a long time because the radioactive bands had to b exposed over time 3) it could only do a small section of dna
how does fluorescent labeling work
the different ddNTP all were tagged with diff. fluorescent colors
