IDENTIFICATION OF FACTOR-IX MUTATIONS IN HEMOPHILIA-B - APPLICATION OF POLYMERASE CHAIN-REACTION AND SINGLE-STRAND CONFORMATION ANALYSIS

被引:14
作者
FRASER, BM
POON, MC
HOAR, DI
机构
[1] UNIV CALGARY,FOOTHILLS HOSP,DEPT MED,1403 29TH ST NW,CALGARY T2N 2T9,ALBERTA,CANADA
[2] CANADIAN RED CROSS,CALGARY,ALBERTA,CANADA
[3] ALBERTA CHILDRENS PROV GEN HOSP,CALGARY T2T 5C7,ALBERTA,CANADA
[4] UNIV CALGARY,ALBERTA CHILDRENS HOSP,RES INST,DEPT MED BIOCHEM,CALGARY T2N 1N4,ALBERTA,CANADA
[5] UNIV CALGARY,ALBERTA CHILDRENS HOSP,RES INST,DEPT PEDIAT,CALGARY T2N 1N4,ALBERTA,CANADA
[6] UNIV CALGARY,FOOTHILLS HOSP,DEPT PEDIAT,CALGARY T2N 1N4,ALBERTA,CANADA
[7] UNIV CALGARY,FOOTHILLS HOSP,DEPT LAB MED,CALGARY T2N 1N4,ALBERTA,CANADA
关键词
D O I
10.1007/BF00215677
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
The molecular characterization of two haemophilia B defects, Calgary 1 and Calgary 2, was carried out using polymerase chain reaction (PCR) amplification and direct dideoxy sequencing. It had been previously shown that the Calgary 1 mutation affects the 5' TaqI restriction site of exon VIII, whereas Calgary 2 involves the loss of the 3' TaqI site of exon VIII of the factor IX gene. Sequencing data has now revealed that each of these alterations involves a C-to-T transition within a CpG dinucleotide. In each instance an arginine residue is replaced by a stop codon. These cases represent the recurrence of each particular alteration, both of which are predicted to result in the production of a truncated protein lacking a significant part of the catalytic region. A recently developed technique that reveals base substitutions as single-strand conformation polymorphisms (SSCP) was adapted for modelling in the detection of point mutations. Referred to here as single-strand conformation (SSC) analysis, this procedure, used in association with PCR, provided a reliable and sensitive system for molecular diagnosis in each of the cases presented. Computer-generated secondary structure predictions demonstrated a strong correlation with experimental results and the technique was used to screen 11 additional patients in the same region. A change detected by SSC analysis in one patient was localized to 55 base pairs, sequenced, and identified as a conservative amino acid substitution. This patient is now referred to as Calgary 3.
引用
收藏
页码:426 / 430
页数:5
相关论文
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