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Monday, 26 November 2007

Genetic engineering (7)

Polymerase and exonuclease
E coli DNA polymerase I
1 chain polypeptide
109000 Dalton
5'->3' polymerase
5'->3" exonuclease
3'-> 5" exonuclease
proof reading and repair DNA
nick translation
DNA labeling- low concentration of DNAse, add label

Klenow fragment,large fragment
1 chain polypeptide
Pol I--> typsin, subtilisin 76000 Dalton
C-terminal, no 5'->3' exonuclease
use for DNA synthesis from RNA, enzymatic sequencing, add nucleotide for 5 protruding end, replacement or exchange reaction of 3' blut end

T4 DNA polymerase
same as Klenow activity, same use
but 3'->5' activity 200X
dNTP concentration control polymerase activity

RNA dependent DNA polymerase, reverse transcriptase
RNA virus enzyme
5'->3' DNA synthesis from RNA template
need ssRNA, or ssDNA as template and primer
make cDNA from mRNA, 3' fill in

Polymerase Chain Reaction (PCR)
  • Template with known at least head or end sequence
  • Design small oligonucletide complement with 3' of each end (20-35 b)
  • Mix large amount of primers with template DNA
  • Deanneal template with heat
  • Reanneal of primer to template
  • DNA polymerase will extend DNA 5'->3'
  • double each round

Taq DNA polymerase - heat stable

usually temperature setting

deanneal-95 1 min

anneal-60 1 min

extension-72 1 min

30-40 rounds

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