Gene walking using sequential hybrid primer polymerase chain reaction

被引：7

作者：

Martin-Harris, Michael H. ^{[1
]}

Bartley, Paul A. ^{[1
]}

Morley, Alexander A. ^{[1
,2
]}

机构：

[1] Flinders Univ & Med Ctr, Dept Hematol & Genet Pathol, Bedford Pk, SA 5042, Australia

[2] Monoquant, Adelaide, SA 5000, Australia

来源：

ANALYTICAL BIOCHEMISTRY | 2010年 / 399卷 / 02期

关键词：

UNKNOWN FLANKING SEQUENCES; ASYMMETRIC INTERLACED PCR; AMPLIFICATION; REGIONS;

D O I：

10.1016/j.ab.2010.01.005

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

We developed a simple and robust method for removing nonspecific amplification produced during gene walking with a gene-specific primer and a degenerate primer. The primary walking polymerase chain reaction (PCR) was followed by two or three PCR rounds, each incorporating a low concentration of a reverse hybrid primer, where the 3' end was bound to a target sequence generated in the preceding PCR round and the 5' end was a new sequence that generated a target sequence for the next PCR round. The low concentration of the hybrid primer and the extent of amplicon stem-loop formation inhibited nonspecific amplification and enabled successful walking along three genes. (c) 2010 Elsevier Inc. All rights reserved.

引用

页码：308 / 310

页数：3

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