Allele frequency ofHinfI restriction fragment length polymorphism (RFLP) and dinucleotide repeat polymorphism in the wilms tumor gene (WT1) among the Japanese

Abstract

The frequency of HinfI RFLP and dinucleotide (CA) repeat polymorphism for the WT1 located to chromosome l lp13 among the Japanese were estimated. Each polymorphism showed high frequency of heterozygosity and segregated independently. Key Words polymorphisms, WT1 gene DNA from 50 Japanese individuals (25 males and 25 females) was amplified by polymerase chain reaction (PCR) followed by Hinfl and/or DraI digestion and electrophoresis.Primers for PCR. A primer set designed by Hoban and Kelsey (199l) as WTII: 5'-GCCTGGAAGAGTTGGTCTCT-3', and WTI2: 5'-ACACAGTAATTTCAAG-CAACGG-3'.Condition of PCR. 100 ng of genomic DNA was amplified with 50 pmol of each primer in 50 t~l PCR reaction mixture (10 mM Tris-C1, pH 8.4/1.5 mM MgCl~/50 mM KC1/250 ~M of each dNTPs/3 units of Taq polymerase). Denaturation at 94~ for 1 rain, annealing at 60~ for 1 rain, and extention at 72~ for 2 min for 30 cycles. PCR products were electrophoresed on 6% polyacrylamide gel (PAG) in 1 x TBE buffer, then stained with ethidium bromide. 1) HinfI RFLP Polymorphie and constant DNA fragments. A two-allele polymorphism with 240 bp (allele a) and 131 bp+ 109 bp (allele b), and three constant fragments (380 bp, 293 bp, and 41 bp) as described by Hobart and Kelsey (1991).Allele frequency. 0.39 for allele "a" and 0.61 for allele "b". PIC=0.36. The frequency of expected and observed heterozygosity was 0.48 and 0.44, respectively.2) CA repeat polymorphism detected after Dral digestion (Fig. 1) Polymorphie and constant DNA fragments.

Molecular analysis of a patient with Beckwith-Wiedemann syndrome, rhabdomyosarcoma and renal cell carcinoma

“…Bell), c-H-ras (from JCRB) and DllS12 (kindly given by Dr. Y. Nakamura). Another part of DNA was used for a polymerase chain reaction (PCR) assay to detect microsatellite nucleotide repeat polymorphism and restriction fragment length polymorphisms (RFLPs) for TH (Human Gene Mapping 11,1991), IGF2 (Ogawa et al, 1993a), HI9 (Rainier et al, 1993;Matsumoto et aL, 1994a), the Wilms tumor suppressor gene (WT1) (Zhang and Tycko, 1992;Matsumoto et al, 1994b) and DllS527 (Human Gene Mapping 11, 1991). Each primer was synthesized by Gene Assembler Plus (Pharmacia LKB).…”