Fork PCR: a universal and efficient genome-walking tool

被引:6
|
作者
Pan, Hao [1 ,2 ,3 ]
Guo, Xinyue [2 ,3 ]
Pan, Zhenkang [2 ,3 ]
Wang, Rongrong [2 ,3 ]
Tian, Bingkun [2 ,3 ]
Li, Haixing [2 ,3 ]
机构
[1] Nanchang Univ, Sch Chem & Chem Engn, Nanchang, Peoples R China
[2] Nanchang Univ, State Key Lab Food Sci & Resources, Nanchang, Peoples R China
[3] Nanchang Univ, Sino German Joint Res Inst, Nanchang, Peoples R China
基金
中国国家自然科学基金;
关键词
genome-walking; fork primer set; primer overlap; randomly partial annealing; nested PCR; ENVIRONMENTAL DNA; INVERSE PCR; AMPLIFICATION; CLONING; SEQUENCES; PROMOTER; SCHEME; GENES;
D O I
10.3389/fmicb.2023.1265580
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The reported genome-walking methods still suffer from some deficiencies, such as cumbersome experimental steps, short target amplicon, or deep background. Here, a simple and practical fork PCR was proposed for genome-walking. The fork PCR employs a fork primer set of three random oligomers to implement walking task. In primary fork PCR, the low-stringency amplification cycle mediates the random binding of primary fork primer to some places on genome, producing a batch of single-stranded DNAs. In the subsequent high-stringency amplification, the target single-strand is processed into double-strand by the site-specific primer, but a non-target single-stranded DNA cannot be processed by any primer. As a result, only the target DNA can be exponentially amplified in the remaining high-stringency cycles. Secondary/tertiary nested fork PCR(s) further magnifies the amplification difference between the both DNAs by selectively enriching target DNA. The applicability of fork PCR was validated by walking several gene loci. The fork PCR could be a perspective substitution for the existing genome-walking schemes.
引用
收藏
页数:7
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