IR@PKUHSC  > 北京大学临床肿瘤学院
学科主题临床医学
Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography
Liu, MR; Pan, KF; Li, ZF; Wang, Y; Deng, DJ; Zhang, L; Lu, YY
刊名WORLD JOURNAL OF GASTROENTEROLOGY
2002-06-01
8期:3页:426-430
收录类别SCI
文章类型Article
WOS标题词Science & Technology
类目[WOS]Gastroenterology & Hepatology
研究领域[WOS]Gastroenterology & Hepatology
关键词[WOS]SINGLE-NUCLEOTIDE POLYMORPHISMS ; P53 GENE MUTATION ; HEPATOCELLULAR-CARCINOMA ; PRENATAL-DIAGNOSIS ; DHPLC ; CANCERS ; BRCA1 ; IDENTIFICATION ; EFFICIENCY ; EXPRESSION
英文摘要

AIM: To optimize conditions of DHPLC and analyze the effectiveness of various DNA polymerases; on DHPLC resolution, and evaluate the sensitivity of DHPLC in the mutation screening of mitochondrial DNA (mtDNA).

METHODS: Two fragments of 16s gene of mitochondrial DNA (one of them F2 is a mutant fragment) and an A3243G mutated fragment were used to analyze the UV detection limit and determine the minimum percentage of mutant PCR products for DHPLC and evaluate effects of DNA polymerases on resolution of DHPLC. Under the optimal conditions, we analyzed the mtDNA mutations from muscle tissues of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes ( MELAS) and screened blindly for variances in D-loop region of mtDNA from human gastric tumor specimen.

RESULTS: Ten A3243G variants were detected in 12 cases of MELAS, no alterations were detected in controls and these results were consistent with the results obtained by analysis of RFLP with Apal. We also identified 26 D-loop variances in 46 cases of human gastric cancer tissues and 38 alterations in 13 gastric cancer cell lines. The mutation of mtDNA at 80 ng PCR products containing a minimum of 5 % mutant sequences could be detected by using DHPLC with UV detector. Moreover, Ampli-Taq Gold polymerase was equally as good as the proofreading DNA polymerase (e. g., Pfu) in eliminating the false positive produced by Taq DNA polymerases.

CONCLUSION: DHPLC is a powerful, rapid and sensitive mutation screening method for mtDNA. Proofreading DNA polymerase is more suitable for. DHPLC analysis than Taq polymerase.

语种英语
WOS记录号WOS:000176452700008
引用统计
被引频次:23[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/58863
专题北京大学临床肿瘤学院
作者单位Peking Univ, Sch Oncol, Beijing Inst Canc Res, Beijing Lab Mol Oncol, Beijing 100034, Peoples R China
推荐引用方式
GB/T 7714
Liu, MR,Pan, KF,Li, ZF,et al. Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography[J]. WORLD JOURNAL OF GASTROENTEROLOGY,2002,8(3):426-430.
APA Liu, MR.,Pan, KF.,Li, ZF.,Wang, Y.,Deng, DJ.,...&Lu, YY.(2002).Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography.WORLD JOURNAL OF GASTROENTEROLOGY,8(3),426-430.
MLA Liu, MR,et al."Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography".WORLD JOURNAL OF GASTROENTEROLOGY 8.3(2002):426-430.
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