公开(公告)号：US20070298428A1

公开(公告)日：2007-12-27

申请号：US11772622

申请日：2007-07-02

申请人： Qiang Liu ,  Steve Sommer ,  Arthur Riggs

发明人： Qiang Liu ,  Steve Sommer ,  Arthur Riggs

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6827 ,  C12Q1/6858 ,  C12Q2525/186 ,  C12Q2521/319 ,  C12Q2535/125 ,  C12Q2565/301 ,  C12Q2535/131

摘要： A novel method of pyrophosphorolysis activated polymerization (PAP) has been developed. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially for each amplification by using an activatable oligonucleotide P* that has a non-extendible 3′-deoxynucleotide at its 3′ terminus. PAP can be applied for exponential amplification or for linear amplification. PAP can be applied to amplification of a rare allele in admixture with one or more wild-type alleles by using an activatable oligonucleotide P* that is an exact match at its 3′ end for the rare allele but has a mismatch at or near its 3′ terminus for the wild-type allele. PAP is inhibited by a mismatch in the 3′ specific sequence as far as 16 nucleotides away from the 3′ terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild-type allele. Specificity results from both pyrophosphorolysis and polymerization since significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, an extremely rare event. Using genetically engineered DNA polymerases greatly improves the efficiency of PAP.

摘要翻译： 开发了焦磷酸活化聚合（PAP）的新方法。 在PAP中，通过使用在其3'末端具有不可延长的3'-脱氧核苷酸的可活化寡核苷酸P *，通过DNA聚合酶进行焦磷酸分解和聚合反应，进行每次扩增。 PAP可以用于指数放大或线性放大。 PAP可以应用于稀释等位基因与一种或多种野生型等位基因的混合，通过使用可激活的寡核苷酸P *，其在3'末端为罕见等位基因而精确匹配，但在3或3附近具有不匹配 '末端为野生型等位基因。 PAP被3'特异性序列的错配抑制至远离3'末端的16个核苷酸。 PAP可以在野生型等位基因存在的情况下大大增加检测极罕见的突变等位基因的特异性。 焦磷酸分解和聚合的特异性结果，因为显着的非特异性扩增需要由DNA聚合酶引起的错配焦磷酸分解和错配合的组合，这是非常罕见的事件。 使用基因工程DNA聚合酶大大提高了PAP的效率。

公开(公告)号：US20050095608A1

公开(公告)日：2005-05-05

申请号：US10798844

申请日：2004-03-12

申请人： Qiang Liu ,  Steve Sommer ,  Arthur Riggs

发明人： Qiang Liu ,  Steve Sommer ,  Arthur Riggs

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6827 ,  C12Q1/6858 ,  C12Q2525/186 ,  C12Q2521/319 ,  C12Q2535/125 ,  C12Q2565/301 ,  C12Q2535/131

摘要： A novel method of pyrophosphorolysis activated polymerization (PAP) has been developed. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially for each amplification by using an activatable oligonucleotide P* that has a non-extendible 3′-deoxynucleotide at its 3′ terminus. PAP can be applied for exponential amplification or for linear amplification. PAP can be applied to amplification of a rare allele in admixture with one or more wild-type alleles by using an activatable oligonucleotide P* that is an exact match at its 3′ end for the rare allele but has a mismatch at or near its 3′ terminus for the wild-type allele. PAP is inhibited by a mismatch in the 3′ specific sequence as far as 16 nucleotides away from the 3′ terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild-type allele. Specificity results from both pyrophosphorolysis and polymerization since significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, an extremely rare event. Using genetically engineered DNA polymerases greatly improves the efficiency of PAP.

摘要翻译： 开发了焦磷酸活化聚合（PAP）的新方法。 在PAP中，通过使用在其3'末端具有不可延长的3'-脱氧核苷酸的可活化寡核苷酸P *，通过DNA聚合酶进行焦磷酸分解和聚合反应，进行每次扩增。 PAP可以用于指数放大或线性放大。 PAP可以应用于稀释等位基因与一种或多种野生型等位基因的混合，通过使用可激活的寡核苷酸P *，其在3'末端为罕见等位基因而精确匹配，但在3或3附近具有不匹配 '末端为野生型等位基因。 PAP被3'特异性序列的错配抑制至远离3'末端的16个核苷酸。 PAP可以在野生型等位基因存在的情况下大大增加检测极罕见的突变等位基因的特异性。 焦磷酸分解和聚合的特异性结果，因为显着的非特异性扩增需要由DNA聚合酶引起的错配焦磷酸分解和错配合的组合，这是非常罕见的事件。 使用基因工程DNA聚合酶大大提高了PAP的效率。