Journal Article

T‐linker‐specific ligation PCR (T‐linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends

Yan Yuanxin, An Chengcai, Li Li, Gu Jiayu, Tan Guihong and Chen Zhangliang

in Nucleic Acids Research

Volume 31, issue 12, pages e68-e68
Published in print June 2003 | ISSN: 0305-1048
Published online June 2003 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gng068
T‐linker‐specific ligation PCR (T‐linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends

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Dozens of PCR‐based methods are available for chromosome walking from a known sequence to an unknown region. These methods are of three types: inverse PCR, ligation‐mediated PCR and randomly primed PCR. However, none of them has been generally applied for this purpose, because they are either difficult or inefficient. Here we describe a simple and efficient PCR strategy—T‐linker‐specific ligation PCR (T‐linker PCR) for gene or chromosome walking. The strategy amplifies the template molecules in three steps. First, genomic DNA is digested with 3′ overhang enzymes. Secondly, primed by a specific primer, a strand of the target molecule is replicated by Taq DNA polymerase and a single A tail is generated on the 3′ unknown end of the target molecule, and then a 3′ overhang‐T linker (named T‐linker) is specifically ligated onto the target. Thirdly, the target is amplified by two rounds of nested PCR with specific primers and T‐linker primers. T‐linker PCR significantly improves the existing PCR methods for walking because it uses specific T/A ligation instead of arbitrary ligation or random annealing. To show the feasibility and efficiency of T‐linker PCR, we have exploited this method to identify vector DNA or T‐DNA insertions in transgenic plants.

Journal Article.  4114 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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