Manipulation of DNA - Polymerases Flashcards
what are polymerases
polymerases are enzymes that add nucleotides to the ends of a primer or to an upstream nucleotide
what other functions can dna polymerase perform
proof reading and nick filling
what direction does dna polymerase work in
5’ to 3’
what is nick translation
nick translation adds nucleotides to a nicked strand, first by removing the postceeding nucleotides, then filling in
what are klenow fragments
a modified form of dna polymerase that contains 5-3 polymerase, and 3-5 exonuclease but not the other. fills gaps, and can be used to blunt sticky ends, digest overhang or prepare labelled probes
what is reverse transcriptase
reverse transcriptase are a template dependant polymerase, sythesizes a dna strand from rna
what is cdna and how is it made
it is made by reverse transcriptase, using rna, lacks introns, and requires primers such as oligio dts or specific sequences
what is pcr
pcr or polymerase chain reaction, is a method to copy dna
what are the requirements for pcr
a template source of dna
two dna primers, forward and reverse
dna polymerase
dntps
a buffer
what are primers
short strands of non phosphorylated dna that is typically 15-40 bases
can also contain restriction sites
what are the two types of dna polymerase used in pcr
taq and pfu
what are some features of taq polymerase
named for thermus aquaticus
optimal growth 65-70
highly error prone, 1 per 1000
what are some features of pfu polymerase
named for pyrococcous furious
optimal growth 100
high fidelity
what are the three stages of pcr
denaturation
primer anealing
dna polymerase
what are the stages and tempratures of pcr
92 - denaturation
55 - primer anealing
72 - extension
what are the differences in the clonability of different pcr enzymes
taq produces an a overhang, that can be attached to a t tailed vector
pfu produces blunt ends that can be ligated directly
how else can pcr products be cloned
using primers with re on the end allow for ligation after pcr
what is alkaline phosphotase
removes 5’ phosphate groups prevents plasmid recircularisation
what is polynucleotide kinase
it adds a 5’ phosphate
