KTS-01 說明書

PAP (Pyrophosphorolysis-activated polymerization)/ KlenTaq-S

PAP is a unique method for DNA amplification (Liu and Sommer: BioTechniques, 29 [2000], 1072-1080; Nucleic Acids Res, 30 [2002], 598-604), where pyrophosphorolysis and polymerization are serially coupled by DNA polymerase using 3’ blocked primer.  Pyrophosphorolysis is the reverse reaction of DNA polymerization where the blocked nucleotide (generally dideoxy) at the 3’ end of the primer template duplex is removed by the enzyme in presence of inorganic pyrophosphate.  After the removal of the blocked nucleotide by pyrophosphorolysis, the activated primer can then be extended by DNA polymerization.

Because of the high specificity (1 / 3x10¹¹) of PAP, this process is becoming a powerful tool for detecting rare mutations, large heterozygous deletions, gene duplications etc in the presence of a large excess of wild type allele (Liu et al : Biotechniques, 40[2006], 661-668).  In theory PAP can detect a single base mutation in 3x10¹¹ copies of wild type allele.

PAP can be used as an alternative or to substantiate the Real Time PCR (Boon et al : Prenat  Diagn 27[2007], 932-937)

KlenTaq-S and PAP reaction.

KlenTaq-S has been found to be a suitable thermostable Taq DNA polymerase by many research groups in PAP reactions.

KlenTaq-S is expressed from 5’ deletion and F667Y (Tabor and Richardson) mutation of the gene, encoding Thermus aquaticus DNA polymerase. Repeated exposure to 98⁰C in the PR1 reaction buffer the enzyme does not show any noticeable loss of activity.

Concentration of KlenTaq-S :      10 U/µl

Suggested Reaction Conditions for PAP (in 25 µl) :

50 mM Tris-HCl, 7.8, 16 mM Ammonium sulfate, 3.5 mM Magnesium chloride,  25 µM each four dNTPs, 0.1 µM each primer, 5-200 ng template, 90 µM inorganic pyrophosphate (Na₄PPi), 2% dimethylsulfoxide (DMSO), 2.5 U of KlenTaq-S.

Cycling Conditions :

95⁰C  for 1 min (denaturation), then

94⁰C for 15 sec

60⁰C for 30 sec

64⁰C for 30 sec

68⁰C for 1 min

72⁰C for 1-2 min      total of 25-45 cycles

Buffer supplied  (10X PR1):

500 mM Tris-HCl, pH 7.8, 160 mM  (NH₄)₂SO₄, 35 mM MgCl₂, 1.5 mg/ml BSA

Ordering Information :

Order No. :                         KTS-01

Quantity  :                           100 µl or 1000 U/vial

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