Variations among Japanese of the factor IX gene (F9) detected by PCR-denaturing gradient gel electrophoresis.
In the course of feasibility studies to examine the efficiencies and practicalities of various techniques for screening for genetic variations, the human coagulation factor IX (F9) genes of 63 Japanese families were examined by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Four target sequences with lengths of 983-2,891 bp from the F9 genes of 126 unrelated individuals from Hiroshima and their 100 children were amplified by PCR, digested with restriction enzymes to approximately 500-bp fragments, and examined by DGGE--a total of 6,724 bp being examined per individual. GC-rich sequences (GC-clamps) of 40 bp were attached to both ends of the target sequences, as far as was feasible. Eleven types of new nucleotide substitutions were detected in the population, none of which produced RFLPs or caused hemophilia B. By examining two target sequences in a single lane, approximately 8,000 bp in a diploid individual could be examined. This approach is very effective for the detection of variations in DNA and is applicable to large-scale population studies.
Satoh
C
C
Department of Genetics, Radiation Effects Research Foundation, Hiroshima, Japan.
Takahashi
N
N
Asakawa
J
J
Hiyama
K
K
Kodaira
M
M
D001483:Base Sequence; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005164:Factor IX / Q000235:genetics*; D005240:Feasibility Studies; D005260:Female; D014644:Genetic Variation; D006801:Humans; D007564:Japan; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D004277:DNA, Single-Stranded; D005164:Factor IX