|
Download
protocol
53kb
pdf? |
|
Abstract for DNA Polymerase I Large (Klenow) Fragment
DNA Polymerase I Large (Klenow) Fragment consists of a single polypeptide chain (68kDa)
that lacks the 5´->3´ exonuclease activity of intact E. coli DNA polymerase
I, but retains its 5´->3´ polymerase, 3´->5´ exonuclease and strand displacement
activities. The 5´->3´ polymerase activity of Klenow Fragment can be used in
the following applications: a) to fill in 5´-protruding ends with unlabeled or labeled dNTPs; b) to sequence single or double-stranded DNA templates;
c) for in vitro
mutagenesis experiments using synthetic oligonucleotides; d) for cDNA second strand
synthesis; and e) to generate single-stranded DNA probes.
The 3´->5´ exonuclease
activity can be used to generate blunt ends from a
3´-overhang. Klenow Fragment,
Exonuclease Minus, which is deficient in both
the 5´->3´ and the 3´->5´ exonuclease activities, is primarily used for sequencing of DNA templates.
|
