{"title":"Primer extension refractory PCR: an efficient and reliable genome walking method","authors":"Haixing Li, Zhiyu Lin, Xinyue Guo, Zhenkang Pan, Hao Pan, Dongying Wang","doi":"10.1007/s00438-024-02126-5","DOIUrl":null,"url":null,"abstract":"<p>Genome walking, a molecular technique for obtaining unknown flanking genomic sequences from a known genomic sequence, has been broadly applied to determine transgenic sites, mine new genetic resources, and fill in chromosomal gaps. This technique has advanced genomics, genetics, and related disciplines. Here, an efficient and reliable genome walking technique, called primer extension refractory PCR (PER-PCR), is presented. PER-PCR uses a set of primary, secondary, and tertiary walking primers. The middle 15 nt of the primary walking primer overlaps with the 3′ parts of the secondary and tertiary primers. The 5′ parts of the three primers are heterologous to each other. The short overlap allows the walking primer to anneal to its predecessor only in a relaxed-stringency PCR cycle, resulting in a series of single-stranded DNAs; however, the heterologous 5′ part prevents the creation of a perfect binding site for the walking primer. In the next stringent cycle, the target single strand can be extended into a double-stranded DNA molecule by the sequence-specific primer and thus can be exponentially amplified by the remaining stringent cycles. The nontarget single strand fails to be enriched due to the lack of a perfect binding site for any primer. PER-PCR was validated by extension into unknown flanking regions of the <i>hyg</i> gene in rice and the <i>gadR</i> gene in <i>Levilactobacillus brevis</i> CD0817. In summary, in this study, a new practical PER-PCR method was constructed as a potential alternative to existing genome walking methods.</p>","PeriodicalId":18816,"journal":{"name":"Molecular Genetics and Genomics","volume":null,"pages":null},"PeriodicalIF":2.3000,"publicationDate":"2024-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Genetics and Genomics","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s00438-024-02126-5","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Genome walking, a molecular technique for obtaining unknown flanking genomic sequences from a known genomic sequence, has been broadly applied to determine transgenic sites, mine new genetic resources, and fill in chromosomal gaps. This technique has advanced genomics, genetics, and related disciplines. Here, an efficient and reliable genome walking technique, called primer extension refractory PCR (PER-PCR), is presented. PER-PCR uses a set of primary, secondary, and tertiary walking primers. The middle 15 nt of the primary walking primer overlaps with the 3′ parts of the secondary and tertiary primers. The 5′ parts of the three primers are heterologous to each other. The short overlap allows the walking primer to anneal to its predecessor only in a relaxed-stringency PCR cycle, resulting in a series of single-stranded DNAs; however, the heterologous 5′ part prevents the creation of a perfect binding site for the walking primer. In the next stringent cycle, the target single strand can be extended into a double-stranded DNA molecule by the sequence-specific primer and thus can be exponentially amplified by the remaining stringent cycles. The nontarget single strand fails to be enriched due to the lack of a perfect binding site for any primer. PER-PCR was validated by extension into unknown flanking regions of the hyg gene in rice and the gadR gene in Levilactobacillus brevis CD0817. In summary, in this study, a new practical PER-PCR method was constructed as a potential alternative to existing genome walking methods.
期刊介绍:
Molecular Genetics and Genomics (MGG) publishes peer-reviewed articles covering all areas of genetics and genomics. Any approach to the study of genes and genomes is considered, be it experimental, theoretical or synthetic. MGG publishes research on all organisms that is of broad interest to those working in the fields of genetics, genomics, biology, medicine and biotechnology.
The journal investigates a broad range of topics, including these from recent issues: mechanisms for extending longevity in a variety of organisms; screening of yeast metal homeostasis genes involved in mitochondrial functions; molecular mapping of cultivar-specific avirulence genes in the rice blast fungus and more.