Molecular haplotyping of genomic DNA for multiple single-nucleotide polymorphisms located kilobases apart using long-range polymerase chain reaction and intramolecular ligation.
Genetic polymorphisms are well-recognized causes of interindividual differences in disease risk and treatment response in humans. For genes containing multiple single-nucleotide polymorphisms (SNPs), haplotype structure is often the principal determinant of phenotypic consequences, and haplotype distribution represents the best approach for assessing patterns of linkage disequilibrium. To permit more widespread molecular determination of haplotypes, we developed a simple yet robust method to determine haplotype structure for multiple SNPs located up to 30 kb apart in genomic DNA using long-range polymerase chain reaction (LR-PCR) and intramolecular ligation. Complete concordance was shown between the new method and conventional approaches, such as family pedigree analysis or cloning and sequencing. The availability of a simple method to directly determine haplotype structure using genomic DNA, without family pedigree analysis, cloning or complex instrumentation, provides an important new tool for elucidating the genetic determinants of drug disposition and effects, disease risk, and molecular evolution.
McDonald
Oliver G
OG
St Jude Children's Research Hospital, Memphis, TN 38101-0318, USA.
Krynetski
Eugene Y
EY
Evans
William E
WE
D000483:Alleles; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D006239:Haplotypes / Q000235:genetics*; D006801:Humans; D008026:Ligation; D008780:Methyltransferases / Q000235:genetics*; D006899:Mixed Function Oxygenases / Q000235:genetics*; D010375:Pedigree; D010597:Pharmacogenetics; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D019948:Receptors, Interleukin-4 / Q000235:genetics*; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D019948:Receptors, Interleukin-4; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D008780:Methyltransferases; C022745:thiopurine methyltransferase