Primer extension refractory PCR: an efficient and reliable genome walking method

IF 2.3 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Haixing Li, Zhiyu Lin, Xinyue Guo, Zhenkang Pan, Hao Pan, Dongying Wang
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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.

Abstract Image

引物延伸耐受性 PCR:一种高效可靠的基因组行走方法
基因组走查是一种从已知基因组序列获取未知侧翼基因组序列的分子技术,已被广泛应用于确定转基因位点、挖掘新的基因资源和填补染色体空白。这项技术推动了基因组学、遗传学和相关学科的发展。本文介绍了一种高效可靠的基因组走查技术,即引物延伸耐受性 PCR(PER-PCR)。PER-PCR 使用一组一级、二级和三级走位引物。主引物中间的 15 nt 与二级和三级引物的 3′部分重叠。这三种引物的 5′部分相互异源。短的重叠部分使得走行引物只能在松弛的 PCR 循环中与其前引物退火,从而产生一系列单链 DNA;但是,异源的 5′部分阻碍了走行引物形成完美的结合位点。在下一个严格循环中,目标单链可通过序列特异性引物扩展成双链 DNA 分子,从而在剩余的严格循环中进行指数扩增。由于任何引物都缺乏完美的结合位点,因此非目标单链无法富集。通过延伸到水稻 hyg 基因和 Levilactobacillus brevis CD0817 的 gadR 基因的未知侧翼区域,PER-PCR 得到了验证。总之,本研究构建了一种新的实用 PER-PCR 方法,有可能替代现有的基因组行走方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Molecular Genetics and Genomics
Molecular Genetics and Genomics 生物-生化与分子生物学
CiteScore
5.10
自引率
3.20%
发文量
134
审稿时长
1 months
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.
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