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

我們公司zui大優(yōu)勢(shì)是強(qiáng)大的采購(gòu)，

1：基本什么都能進(jìn)口，血清，抗體，耗材，還有部分限制進(jìn)口的，

2：貨品全，現(xiàn)經(jīng)營(yíng)過(guò)700多個(gè)品牌，基本所有生物試劑耗材都可以進(jìn)口，特別是冷偏的產(chǎn)品那就更有優(yōu)勢(shì)，

3：提供加急服務(wù)，一般1-2周到貨，超過(guò)時(shí)限加急費(fèi)全免

4：價(jià)格公道，絕大部分價(jià)格有優(yōu)勢(shì)，當(dāng)然不能保證100%產(chǎn)品都是,因?yàn)橐馕吨鴽](méi)有服務(wù).

5：良好的信譽(yù)，大部分客戶(hù)我們提供貨到付款服務(wù)，客戶(hù)包括清華，北大 交大 復(fù)旦，中山等100多所大學(xué)，ROCHE，阿斯利康，國(guó)藥 ，fisher等500多家公司

6：我們*代理的品牌有：Antibody Research Corporation，arcticzymes，Biorelevant，AmberGen, Inc. ，clemente-associates，clodronaiposomes，Columbia Biosciences，enzyme research，Gene Bridges GmbH，Genovis，AmberGen, Inc.  Biotechnology GmbH，Haematologic Technologies HTI Haemtech，hookelabs，Immudex，Innovative Research of America，inspiralis ，List Biological Laboratories, Inc.，lumafluor，Microsurfaces，multiplicom，nanotools，Pel-Freez Biologicals，pentapharm，progen，Protein Ark，QA-Bio,Inc，QA-Bio,IncQuickZyme Biosciences，Teknova，TriLink BioTechnologies, Inc.，Zyagen Laboratories 等

7：我們還是invitrogen,qiagen,Midland BioProducts Corporationam,sigma;neb,roche,merck, rnd,BD, GE,pierce,BioLegend等知名*批發(fā)，歡迎合作。