Tm-shift基因分型方法在遗传学中的应用

doi:10.3724/SP.J.1005.2012.01484

遗传 ›› 2012, Vol. 34 ›› Issue (11): 1484-1490.doi: 10.3724/SP.J.1005.2012.01484

Tm-shift基因分型方法在遗传学中的应用

袁芳¹, 徐进^1,2, 季林丹^1,2, 费丽娟¹, 刘盼盼¹, 张莉娜¹

1. 宁波大学医学院, 宁波 315211 2. 中国科学院昆明动物研究所-香港中文大学生物资源与疾病分子机理联合实验室, 昆明 650223

收稿日期:2012-08-27 修回日期:2012-10-10 出版日期:2012-11-20 发布日期:2012-11-25
通讯作者: 张莉娜 E-mail:zhanglina@nbu.edu.cn
基金资助:
浙江省自然科学基金项目(编号：(Y2100857))、浙江省教育厅重点科研项目(编号：Z201017918)和宁波市自然科学基金项目(编号：2012A610237, 2011A610037)资助

Application of Tm-shift genotyping method in genetic studies

YUAN Fang¹, XU Jin^1,2, JI Lin-Dan^1,2, FEI Li-Juan¹, LIU Pan-Pan¹, ZHANG Li-Na¹

1. Department of Preventive Medicine, School of Medicine, Ningbo University, Ningbo 315211, China 2. KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research of Common Diseases, Kunming 650223, China

Received:2012-08-27 Revised:2012-10-10 Online:2012-11-20 Published:2012-11-25

摘要/Abstract

摘要： Melting Temperature shift(Tm-shift)是一种新的基因分型方法, 主要通过在两条特异性引物5′端加入不同长度的GC序列, PCR扩增后根据熔解曲线中产物Tm值的差异来完成分型。文章建立了Tm-shift法对2 048份样品的29个SNP进行分型, 通过分型成功率、重复检测一致率、测序验证准确度综合评价分型效果。结果显示, 29个SNP中有27个可以采用本方法分型, 分型成功率为93.1%。测序验证准确性达到100%。3种基因型阳性标准对照重复检测一致率为100%; 100个随机样品重复检测, 重复性为97%。因此, Tm-shift基因分型法是一种成本低廉、准确灵敏、稳定可靠、通量灵活、操作简便的基因分型方法, 可在遗传学研究中推广应用。

关键词: Tm-shift, 基因分型, 遗传学

Abstract: Melting Temperature shift (Tm-shift) is a new genotyping method. With two GC-rich tails of unequal length combined to 5′-terminal of allele-specific primers, genotypes can be determined by the distinct Tms of the PCR products with inspection of a melting curve on the real-time PCR machine. In this study, 29 SNPs were genotyped with 2 048 samples by using Tm-shift genotyping method, and the results were assessed by success rate, consistent rate, and accuracy. The results indicated that among 29 SNPs, 27 SNPs could be genotyped by Tm-shift. In other words, the success rate was 93.1%. The accuracy confirmed by direct sequencing was 100%. The consistency was 100% with 3 control samples, and 97% from a replication study in 100 samples. Thus, Tm-shift is a genotyping method with advantages including low cost, high accuracy, stability, reliability, flexible throughput, and easy operation, which can be applied to genetic studies widely.

Key words: Tm-shift, genotyping, genetics

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[J]. 遗传, 2012, 34(11): 1484-1490.

YUAN Fang XU Jin JI Lin-Dan FEI Li-Juan LIU Pan-Pan ZHANG Li-Na. Application of Tm-shift genotyping method in genetic studies[J]. HEREDITAS, 2012, 34(11): 1484-1490.

参考文献

[1] Shastry BS. SNPs in disease gene mapping, medicinal drug development and evolution. J Hum Genet, 2007, 52(11): 871-880.
[2] Germer S, Higuchi R. Single-tube genotyping without oligonucleotide probes. Genome Res, 1999, 9(1): 72-78.
[3] Wang J, Chuang K, Ahluwalia M, Patel S, Umblas N, Mirel D, Higuchi R, Germer S. High-throughput SNP genotyping by single-tube PCR with Tm-shift primers. Biotechniques, 2005, 39(6): 885-893.
[4] Cardozo GP, Santos EV, Fachin AL, Franca SC, Marins M. A glass bead protocol for recovery of host cell free Herlichia canis and quantification by Sybr-green real-time PCR. Biocell, 2011, 35(1): 35-36.
[5] Gupta M, Yates CR, Meibohm B. SYBR Green-based real- time PCR allelic discrimination assay for beta2-adrenergic receptor polymorphisms. Anal Biochem, 2005, 344(2): 292-294.
[6] Ponchel F, Toomes C, Bransfield K, Leong FT, Douglas SH, Field SL, Bell SM, Combaret V, Puisieux A, Mighell AJ, Robinson PA, Inglehearn CF, Isaacs JD, Markham AF. Real-time PCR based on SYBR-Green I fluorescence: an alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnol, 2003, 3: 18.
[7] Papp AC, Pinsonneault JK, Cooke G, Sadée W. Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves. Biotechniques, 2003, 34(5): 1068-1072.
[8] Reed GH, Kent JO, Wittwer CT. High-resolution DNA melting analysis for simple and efficient molecular diag-nostics. Pharmacogenomics, 2007, 8(6): 597-608.
[9] Birch DE, Kolmodin L, Wong J, Zangenberg GA, Zoccoli MA, McKinney N, Young KKY. Simplified hot start PCR. Nature, 1996, 381(6581): 445-446.
[10] Germer S, Holland MJ, Higuchi R. High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR. Genome Res, 2000, 10(2): 258-266.
[11] Lawyer FC, Stoffel S, Saiki RK, Chang SY, Landre PA, Abramson RD, Gelfand DH. High-level expression, puri-fication, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease activity. PCR Methods Appl, 1993, 2(4): 275-287.
[12] Hsia K, Spector D, Lawrie J, Spector SA. Enzymatic amplification of human cytomegalovirus sequences by polymerase chain reaction. J Clin Microbiol, 1989, 27(8): 1802-1809.
[13] Huang SW, Li Q, Zhu SY, Li L, Xiong F, Jia YK, Xu XM. SYBR Green-based real-time PCR assay for detection of VKORC1 and CYP2C9 polymorphisms that modulate warfarin dose requirement. Clin Chem Lab Med, 2009, 47(1): 26-31.
[14] Tang NL, Liao CD, Ching JK, Suen EW, Chan IH, Orwoll E, Ho SC, Chan FW, Kwok AW, Kwok T, Woo J, Leung PC. Sex-specific effect of Pirin gene on bone mineral density in a cohort of 4000 Chinese. Bone, 2010, 46(2): 543-550.
[15] Chen HY, Chan IHS, Sham AL, Leung VHK, Ma SL, Ho SC, Tang NLS. Haplotype effect in the IGF1 promoter accounts for the association between microsatellite and serum IGF1 concentration. Clin Endocrinol, 2011, 74(4): 520-527.
[16] Derzelle S, Mendy C, Laroche S, Madani N. Use of high- resolution melting and melting temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination. J Microbiol Meth, 2011, 87(2): 195-201.
[17] Liang KH, Fen JJ, Chang HH, Wang HW, Hwang YC. A base-calling algorithm for Tm-shifted melting curve SNP assay. J Clin Bioinforma, 2011, 1(1): 3-8.
[18] Zhou SH, Liu M, An WX, Liang XH, Yu WJ, Gong BL, Piao FY. Genotyping of human platelet antigen-15 by single closed-tube Tm-shift method. Int J Lab Hematol, 2012, 34(1): 41-46.
[19] Tang NL, Chan CY, Leung CC, Tam CM, Blackwell J. Tuberculosis susceptibility genes in the chemokine cluster region of chromosome 17 i

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

Tm-shift基因分型方法在遗传学中的应用

Application of Tm-shift genotyping method in genetic studies

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	马捷, 黄露杰, 张巧霞, 朱艳, 钱露. 以A2型短指(趾)症为案例的医学遗传学PBL教学设计[J]. 遗传, 2023, 45(2): 176-183.
[2]	谢甜, 王梅, 高瑞钰, 苗艳尼, 张燚铭, 蒋婧. 光控诱导重组系统的开发与应用[J]. 遗传, 2022, 44(8): 655-671.
[3]	王飞, 王萌, 张兴华, 宇克莉, 郑连斌, 杨亚军. 中国西南地区3个隔离人群遗传亚结构分析[J]. 遗传, 2022, 44(5): 424-431.
[4]	孔永强, 刘金凯, 顾佳琪, 徐景怡, 郑雨诺, 魏以梁, 伍少远. 南-北方汉族人、韩国人和日本人遗传划分机器学习模型优化方案[J]. 遗传, 2022, 44(11): 1028-1043.
[5]	郭晓媛, 孙昌春, 薛思瑶, 赵慧, 江丽, 李彩霞. 49AISNP：东亚北方三个族群遗传来源推断[J]. 遗传, 2021, 43(9): 880-889.
[6]	王海燕, 龚志云, 蒋甲福, 周宝良, 娄群峰, 曹清河, 席梦利, 陈佩度, 顾铭洪, 张天真, 陈发棣, 陈劲枫, 李宗芸, 王秀娥. 江苏省植物细胞遗传学研究回顾与展望[J]. 遗传, 2021, 43(5): 397-424.
[7]	文子龙, 赵毅强. 群体遗传学下动物驯化研究进展[J]. 遗传, 2021, 43(3): 226-239.
[8]	张向前, 李楠, 解新明. 表观遗传学综合性实验设计与探讨[J]. 遗传, 2021, 43(12): 1179-1187.
[9]	李茜, 王浩宇, 曹悦岩, 朱强, 舒潘寅, 侯婷芸, 王雨婷, 张霁. 微单倍型遗传标记的法医基因组学研究[J]. 遗传, 2021, 43(10): 962-971.
[10]	刘志勇, 乌日嘎, 李燃, 王蔷薇, 孙宏钰. 法医遗传学研究和鉴定中的伦理问题[J]. 遗传, 2021, 43(10): 994-1002.
[11]	孙小媛, 王一帆, 王韫慧, 蔺佳雨, 李金红, 丘远涛, 方小龙, 孔凡江, 李美娜. 大豆细胞核雄性不育基因研究进展[J]. 遗传, 2021, 43(1): 52-65.
[12]	赵娜, 亓宝, 董芊里, 王晓丽. 普通小麦相关研究进展在遗传学理论教学中的应用[J]. 遗传, 2020, 42(9): 916-925.
[13]	王涛, 梁亮, 郑敏化. 形成性评价与教学反馈在医学遗传学PBL教学中的应用[J]. 遗传, 2020, 42(8): 810-816.
[14]	胡颖楚, 胡豪畅, 林少沂, 陈晓敏. DNA羟甲基化调控动脉粥样硬化的研究进展[J]. 遗传, 2020, 42(7): 632-640.
[15]	王小娟, 钱恩芳, 李悦, 宋正阳, 赵慧, 谢何鑫, 李彩霞, 黄江, 江丽. 中国西南地区藏族人群遗传亚结构研究[J]. 遗传, 2020, 42(6): 565-576.