Direct carrier detection in hemophilia B kindreds: use of modified primers (mutagenic primers) for enzymatic amplification of the factor IX gene.
Rapid direct detection of point mutations in hemophilia B kindreds was performed by analyzing the restriction fragments of the factor IX gene amplified by polymerase chain reaction (PCR). The family members of the two patients, HB 5 and HB 6, whose mutant factor IX gene had previously been defined by sequence analysis, were examined in this study. Since there are no restriction endonucleases available for detecting each mutation directly, we designed modified primers which were substituted one nucleotide near the mutated positions. Following PCR with these primers, new Mbo I or Alu I cleavage sites for normal alleles were created. In each family, the restriction fragment revealed the carriership of each patient's mother. This simple methodology to detect mutations of interest is useful for genetic counseling in sporadic cases of hemophilia B.
Matsushita
T
T
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
Tanimoto
M
M
Yamamoto
K
K
Sugiura
I
I
Takamatsu
J
J
Kamiya
T
T
Saito
H
H
D000483:Alleles; D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific; D005164:Factor IX / Q000032:analysis / Q000235:genetics*; D005260:Female; D006580:Genetic Carrier Screening / Q000379:methods*; D002836:Hemophilia B / Q000175:diagnosis* / Q000235:genetics / Q000517:prevention & control; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
D015345:Oligonucleotide Probes; D005164:Factor IX; C024669:endodeoxyribonuclease AluI; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases