Linear allele-specific long-range amplification: a novel method of long-range molecular haplotyping.
Haplotypes have been repeatedly shown to be more powerful than collections of single-locus markers in gene-mapping studies. Various haplotyping methods including statistical estimation are employed but molecular haplotyping, the acquisition of information directly on physical DNA sequences, has been in demand for its accuracy and independence from family pedigrees. We investigated the allelic specificity of long-range PCR, which was successful for long-range haplotyping in recent reports, and found problems of initial mispriming and crossover amplification significantly confounded its application. Based on these observations, we designed a novel method based on linear amplification of a hemizygous DNA segment with a single phosphorothioate-modified oligonucleotide. Our results revealed, with a single nucleotide polymorphism as the discriminative marker, downstream haplotypes of 14-15 kb DNA segment could be confidently scored. With two rounds of the method and five single nucleotide polymorphisms, molecular haplotypes of 29.3 kb spanning the HCR and CDSN genes, two genes associated with the susceptibility of psoriasis, of 11 members, belonging to a CEPH family, were revealed. Clear Mendelian segregation of 35 highly heterozygous SNPs confirmed the accuracy of the method. Problems of low specificity associated with long-range PCR were not observed. The simplicity, along with long-sequence accessibility and feasibility of a single nucleotide difference as the discriminative marker indicated our method holds promise for future gene-mapping studies.
Wu
Wei-Ming
WM
Department of Dermatology, Chang Gung Memorial Hospital, Taipei, Taiwan. weimin1970@adm.cgmh.org.tw
Tsai
Hsiang-Ju
HJ
Pang
Jong-Hwei S
JH
Wang
Tzu-Hao
TH
Wang
Hsin-Shih
HS
Hong
Hong-Shang
HS
Lee
Yun-Shien
YS
D011125:Adenomatous Polyposis Coli / Q000175:diagnosis / Q000235:genetics; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000483:Alleles; D015179:Colorectal Neoplasms / Q000235:genetics; D045647:DNA Glycosylases / Q000235:genetics; D005260:Female; D017491:Genes, APC; D014644:Genetic Variation; D018095:Germ-Line Mutation; D006239:Haplotypes; D006801:Humans; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010375:Pedigree
D045647:DNA Glycosylases; C061738:mutY adenine glycosylase