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学科主题: 临床医学
题名:
Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography
作者: Liu, MR; Pan, KF; Li, ZF; Wang, Y; Deng, DJ; Zhang, L; Lu, YY
刊名: WORLD JOURNAL OF GASTROENTEROLOGY
发表日期: 2002-06-01
卷: 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
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/58863
Appears in Collections:北京大学临床肿瘤学院_期刊论文

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作者单位: Peking Univ, Sch Oncol, Beijing Inst Canc Res, Beijing Lab Mol Oncol, Beijing 100034, Peoples R China

Recommended Citation:
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.
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