Modification of primers for GRHPR genotyping: avoiding allele dropout by single nucleotide polymorphisms and homology sequence.
Mutation of primer site for genotyping by polymerase chain reaction (PCR) may cause allele dropout and other genotyping failures. Primary hyperoxaluria type 2 (PH2) is a rare inherited disease caused by overproduction of endogenous oxalate due to mutations in the glyoxylate/hydroxypyruvate reductase (GRHPR) gene. Here, to avoid allele dropout and primer annealing to multiple sites, and given the discrepancy in intron length between GRHPR gene data, we updated the primers used in the sequence assay of the GRHPR gene. These redesigned primers show potential in reducing detection failure of GRHPR mutations. In addition, we performed a single nucleotide polymorphism (SNP) linkage analysis of the GRHPR gene using direct sequencing with PCR amplification of specific alleles (DS-PASA). Using this technique, we sequenced four common SNPs between intron E and exon 6, which show linkage disequilibrium (LD) consisting of three types of haplotypes, similar to data from the HapMap SNP database.
Takaoka
Naohisa
N
Department of Urology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka, 431-3192, Japan.
Takayama
Tatsuya
T
Miyazaki
Miki
M
Nagata
Masao
M
Ozono
Seiichiro
S
D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000483:Alleles; D001483:Base Sequence / Q000235:genetics; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005838:Genotype; D006801:Humans; D006960:Hyperoxaluria, Primary / Q000235:genetics; D007438:Introns / Q000235:genetics; D015810:Linkage Disequilibrium / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D004247:DNA; D000429:Alcohol Oxidoreductases; C020830:glyoxylate reductase