低温变性下复合PCR技术及其应用

doi:10.16288/j.yczz.17-369

遗传 ›› 2018, Vol. 40 ›› Issue (3): 227-236.doi: 10.16288/j.yczz.17-369

低温变性下复合PCR技术及其应用

梁卉^1,²,陈国杰³,于燕⁴,熊礼宽^1,²()

^1. 暨南大学附属深圳市宝安区妇幼保健院中心实验室,深圳 518102
^2. 深圳市出生缺陷重点实验室,深圳 518102
^3. 郑州大学附属第一医院消化科,郑州 450052
^4. 暨南大学附属深圳市宝安区妇幼保健院产科,深圳 518102

收稿日期:2017-11-06 修回日期:2018-02-02 出版日期:2018-03-20 发布日期:2018-02-08
基金资助:
深圳市三名工程-出生缺陷防治研究与转化团队(编号：SZSM201406007),深圳市出生缺陷重点实验室(编号：ZDSYS201504301707152)和深圳市宝安区医疗卫生基础研究项目(编号：2014067,2017JD001)资助

Co-amplification at lower denaturation temperature-PCR: methodology and applications

Hui Liang^1,²,Guojie Chen³,Yan Yu⁴,Likuan Xiong^1,²()

^1. Central Laboratory, Bao’an Maternal and Child Health Hospital, Jinan University, Shenzhen 518102, China;
^2. Shenzhen Key Laboratory of Birth Defects, Shenzhen 518102, China
^3. Department of Gastroenterology, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
^4. Department of Obstetrics, Bao’an Maternal and Child Health Hospital, Jinan University, Shenzhen 518102, China;

Received:2017-11-06 Revised:2018-02-02 Online:2018-03-20 Published:2018-02-08
Supported by:
[Supported by Sanming Project of Medicine in Shenzhen-Brith Defects Prevention Research and Transformation Team (No. SZSM201406007), Shenzhen Key Laboratory of Birth Defects (No. ZDSYS201504301707152 and Science and Technology Plan Project of Baoan District (Nos. 2014067, 2017JD001)]

摘要/Abstract

摘要：

低温变性下复合PCR(co-amplification at lower denaturation temperature-polymerase chain reaction, COLD- PCR)是一种在高丰度野生型序列背景下选择性变性和扩增低丰度突变型序列的方法,可将突变型序列富集10~100倍。基于突变片段Tm值的改变和异源双链DNA分子的形成,COLD-PCR可富集扩增片段中所有类型和位置的突变,也可富集未知突变,具有敏感、特异、精确、廉价和易操作等优点。COLD-PCR及其衍生方法被应用于肿瘤、微生物、产前筛查和动植物等领域,对疾病的早期诊断、病程和治疗监控、药物选择、预后判断和植物育种等均有积极的作用。本文就COLD-PCR的原理、关键技术、衍生方法及其应用进行综述。

关键词: 低温变性下复合PCR, 精确的变性温度, 选择性富集, 低丰度突变, 检测阈值

Abstract:

Co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) is a novel form of PCR that selectively denatures and amplifies low-abundance mutations from mixtures of wild-type and mutation-containing sequences, enriching the mutation 10 to 100 folds. Due to the slightly altered melting temperature (Tm) of the double-stranded DNA and the formation of the mutation/wild-type heteroduplex DNA, COLD-PCR methods are sensitive, specific, accurate, cost-effective and easy to maneuver, and can enrich mutations of any type and at any position, even unknown mutations within amplicons. COLD-PCR and its improved methods are now applied in cancer, microorganisms, prenatal screening, animals and plants. They are extremely useful for early diagnosis, monitoring the prognosis of disease and the efficiency of the treatment, drug selection, prediction of prognosis, plant breeding and etc. In this review, we introduce the principles, key techniques, derived methods and applications of COLD-PCR.

Key words: co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR), Tc, selectively amplifies, low-abundance mutation, detection threshold

梁卉, 陈国杰, 于燕, 熊礼宽. 低温变性下复合PCR技术及其应用[J]. 遗传, 2018, 40(3): 227-236.

Hui Liang, Guojie Chen, Yan Yu, Likuan Xiong. Co-amplification at lower denaturation temperature-PCR: methodology and applications[J]. Hereditas(Beijing), 2018, 40(3): 227-236.

参考文献

[1]	Lu YW, Wang KH . Research progress on genetic heterogeneity between primary and paired metastatic colorectal cancer. Hereditas (Beijing), 2017,39(6):482-490.
[1]	鲁有望, 王昆华 . 结直肠癌原发与配对转移肿瘤遗传异质性研究进展. 遗传, 2017,39(6):482-490.
[2]	Milbury CA, Li J, Makrigiorgos GM . PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem, 2009,55(4):632-640.
[3]	Li J, Wang LL, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM . Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med, 2008,14(5):579-584.
[4]	How Kit A, Mazaleyrat N, Daunay A, Nielsen HM, Terris B, Tost J . Sensitive detection of KRAS mutations using enhanced-ice-COLD-PCR mutation enrichment and direct sequence identification. Hum Mutat, 2013,34(11):1568-1580.
[5]	Castellanos-Rizaldos E, Richardson K, Lin R, Wu G, Makrigiorgos MG . Single-tube, highly parallel mutation enrichment in cancer gene panels by use of temperature-tolerant COLD-PCR. Clin Chem, 2015,61(1):267-277.
[6]	Yuan F, Xu J, Ji LD, Fei LJ, Liu PP, Zhang LN . Application of Tm-shift genotyping method in genetic studies. Hereditas (Beijing), 2012,34(11):1484-1490.
[6]	袁芳, 徐进, 季林丹, 费丽娟, 刘盼盼, 张莉娜 . Tm- shift基因分型方法在遗传学中的应用. 遗传, 2012,34(11):1484-1490.
[7]	Castellanos-Rizaldos E, Paweletz C, Song C, Oxnard GR, Mamon H, J?nne PA, Makrigiorgos GM . Enhanced ratio of signals enables digital mutation scanning for rare allele detection. J Mol Diagn, 2015,17(3):284-292.
[8]	Milbury CA, Correll M, Quackenbush J, Rubio R, Makrigiorgos GM . COLD-PCR enrichment of rare cancer mutations prior to targeted amplicon resequencing. Clin Chem, 2012,58(3):580-589.
[9]	Li J, Milbury CA, Li C, Makrigiorgos GM . Two- round coamplification at lower denaturation temperature-PCR (COLD-PCR)-based sanger sequencing identifies a novel spectrum of low-level mutations in lung adenocarcinoma. Hum Mutat, 2009,30(11):1583-1590.
[10]	Kristensen LS, Daugaard IL, Christensen M, Hamilton-Dutoit S, Hager H, Hansen LL . Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products. Hum Mutat, 2010,31(12):1366-1373.
[11]	Hashida S, Soh J, Toyooka S, Tanaka T, Furukawa M, Shien K, Yamamoto H, Asano H, Tsukuda K, Hagiwara K, Miyoshi S . Presence of the minor EGFR T790M mutation is associated with drug-sensitive EGFR mutations in lung adenocarcinoma patients. Oncol Rep, 2014,32(1):145-152.
[12]	Milbury CA, Li J, Makrigiorgos GM . Ice-COLD-PCR enables rapid amplification and robust enrichment for low- abundance unknown DNA mutations. Nucleic Acids Res

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低温变性下复合PCR技术及其应用

Co-amplification at lower denaturation temperature-PCR: methodology and applications

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