A primer-based approach to genome walking

被引：5

作者：

Ashoub, Ahmed ^{[1
]}

Abdalla, Khaled S. ^{[1
]}

机构：

[1] Agr Res Ctr, Agr Genet Engn Res Inst, Giza 12619, Egypt

来源：

PLANT MOLECULAR BIOLOGY REPORTER | 2006年 / 24卷 / 02期

关键词：

genome walking; 3 ' overhangs; primer ligation; restriction endonucleases; single stranded DNA primers;

D O I：

10.1007/BF02914062

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A genome walking strategy based on annealing and ligation of single-stranded DNA primers to 3' overhangs following restriction endonuclease digestion was developed. A set of primers contains 4 nucleotides at the 3' end that are complementary to overhangs formed by restriction endonucleases ApaI; PstI; SacI and SphI. Following ligation, 5' end overhangs are formed on the DNA, which serves as sites for the adaptor primers and nested primers for PCR amplification in combination with the gene-specific primers. This strategy was verified by the amplification of up to 4 kb of a potato leafroll virus full-length infectious clone. The procedure could be adopted to target any upstream and downstream regions flanking known sequences within the plant genome.

引用

页码：237 / 243

页数：7

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