Quantitative analysis of human DNA sequences by PCR and solid-phase minisequencing

被引:29
作者
Suomalainen, A [1 ]
Syvänen, AC [1 ]
机构
[1] Univ Uppsala, Dept Med Sci, S-75185 Uppsala, Sweden
关键词
quantitative PCR; minisequencing; allele; frequency; heteroplasmy; gene copy numbers; single nucleotidel; polymorphisms;
D O I
10.1385/MB:15:2:123
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Reliable quantification by PCR requires careful experimental design and conditions, often involving sampling of the PCR reactions at different time points or amplifying multiple dilutions of a standard DNA. We describe here an accurate, quantitative and easily automatizable solid-phase method based on competetive PCR. The PCR products are analyzed by solid-phase minisequencing after capture of biotinylated PCR products in streptavidin-coated microtiter wells and single-nucleotide extension of a specific detection primer by a radioactively labelled nucleotide. The results are expressed as numeric cpm-values, and the incorporated label expresses the relative amount of sequence variants in the original template mixture. We have applied the method to determination of allele frequencies in pooled DNA samples, of mitochondrial heteroplasmy, of gene copy numbers, and to forensic DNA analysis.
引用
收藏
页码:123 / 131
页数:9
相关论文
共 20 条
[1]   BIOTINYLATION OF OLIGONUCLEOTIDES USING A WATER-SOLUBLE BIOTIN ESTER [J].
BENGTSTROM, M ;
JUNGELLNORTAMO, A ;
SYVANEN, AC .
NUCLEOSIDES & NUCLEOTIDES, 1990, 9 (01) :123-127
[2]   TRANSCRIPTION OF THE DYSTROPHIN GENE IN HUMAN-MUSCLE AND NON-MUSCLE TISSUES [J].
CHELLY, J ;
KAPLAN, JC ;
MAIRE, P ;
GAUTRON, S ;
KAHN, A .
NATURE, 1988, 333 (6176) :858-860
[3]   ANALYSIS OF CYTOKINE MESSENGER-RNA AND DNA - DETECTION AND QUANTITATION BY COMPETITIVE POLYMERASE CHAIN-REACTION [J].
GILLILAND, G ;
PERRIN, S ;
BLANCHARD, K ;
BUNN, HF .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (07) :2725-2729
[4]  
IHALAINEN J, 1994, BIOTECHNIQUES, V16, P938
[5]  
Ikonen E, 1992, PCR Methods Appl, V1, P234
[6]  
LAAN M, 1995, HUM GENET, V96, P275
[7]  
LIVAK KJ, 1995, PCR METH APPL, V4, P357
[8]   USE OF THE POLYMERASE CHAIN-REACTION IN THE QUANTITATION OF MDR-1 GENE-EXPRESSION [J].
MURPHY, LD ;
HERZOG, CE ;
RUDICK, JB ;
FOJO, AT ;
BATES, SE .
BIOCHEMISTRY, 1990, 29 (45) :10351-10356
[9]   QUANTITATIVE-ANALYSIS OF MDR1 (MULTIDRUG RESISTANCE) GENE-EXPRESSION IN HUMAN TUMORS BY POLYMERASE CHAIN-REACTION [J].
NOONAN, KE ;
BECK, C ;
HOLZMAYER, TA ;
CHIN, JE ;
WUNDER, JS ;
ANDRULIS, IL ;
GAZDAR, AF ;
WILLMAN, CL ;
GRIFFITH, B ;
VONHOFF, DD ;
RONINSON, IB .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (18) :7160-7164
[10]  
Pastinen T, 1996, CLIN CHEM, V42, P1391