Single-molecule analysis for molecular haplotyping

Kwok, Pui‐Yan; Xiao, Ming

doi:10.1002/humu.20020

Cited by 35 publications

(32 citation statements)

References 26 publications

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“…To study SNPs evenly distributed over long distances, DNA fragments containing adjacent SNPs must be generated to retain the phase information. Methods such as single-molecule PCR and polymerase colony and cloning-based systematic haplotyping can be used to analyze linkage of SNPs separated by thousands of nucleotides (7,8,16,17,23). More recently, the polymerase colony method has been used to generate haplotypes by using multiple amplifications from single molecules (23).…”

Section: Discussionmentioning

confidence: 99%

“…Single-molecule PCR has high sensitivity for 1-kb-sized fragments, albeit at lower efficiency (7,8). High throughput and sensitivity can be achieved with pyrosequencing techniques for 100-bp templates (13)(14)(15).…”

Section: Discussionmentioning

confidence: 99%

“…Laboratory methods for phase determination have been developed including single-molecule PCR, sperm typing, allele-specific PCR, pyrosequencing, bacterial artificial chromosome cloning, polymerase colony, construction of somatic cell hybrids, atomic force microscopy, rolling circle amplification, and mass spectrometry (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Although these methods can define SNP haplotypes, not all are suitable for haplotyping the multilocus MHC region because of its extensive polymorphism and uneven distribution of coding variation (www.ebi.ac.uk͞imgt͞hla) and the physical distance between each HLA locus.…”

Section: Geneticsmentioning

confidence: 99%

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Long-range multilocus haplotype phasing of the MHC

Guo

Hood

Malkki

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Haplotypes are a powerful tool for identifying the genetic basis of common complex diseases. Disease-association mapping requires molecular methods for haplotyping biallelic SNP variation and highly complex polymorphisms. We developed a method for phasing HLA-A, HLA-B, and HLA-DRB1 alleles on chromosome 6 in unrelated individuals. This method uses the highly polymorphic HLA-B locus to discriminate the two HLA haplotypes in heterozygous individuals and its ideal location 1.4 Mbp telomeric to HLA-DRB1 and 1.2 Mbp centromeric to HLA-A to capture 2-Mbp-long genomic DNA. Genomic DNA representing a single HLA-B-captured haplotype is genotyped for HLA-A and HLA-DRB1 alleles and linkage to HLA-B is established. Proof of principle was established in a large blinded study of phase-known samples. Availability of an efficient method for MHC haplotype phase determination will facilitate the mapping of causative MHC-resident genes in many human diseases and has the potential to be broadened to other polymorphic gene complexes.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Geneticsmentioning

confidence: 99%

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Long-range multilocus haplotype phasing of the MHC

Guo

Hood

Malkki

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Currently available molecular haplotyping methods are arduous and typically cost-prohibitive. New methods of molecular haplotype determination are under development (11,12).…”

Section: How Can Haplotypes Be Determined?mentioning

confidence: 99%

Haplotype Thinking in Lung Disease

Silverman¹

2007

Proceedings of the American Thoracic Society

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To identify the genetic etiology of a disease of interest, diseaserelated characteristics (phenotypes) are often tested for association with genetic variants (genotypes). Although genetic association studies of single genetic variants have been widely performed, there has been increasing interest in studies of multiple adjacent genetic variants on one chromosome, known as a haplotype. In this review, we will provide background about the origin of haplotypes and why they can be useful in genetic studies; we will discuss approaches to determining haplotypes and performing haplotype-based genetic association studies; and we will compare single variant and haplotype-based approaches.

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“…Conventional genotyping tools cannot generate haplotype information for a diploid cell [Kwok and Xiao, 2004]. For example, a genomic region with four biallelic markers can yield sixteen possible haplotypes.…”

Section: Introductionmentioning

confidence: 99%

Linear allele-specific long-range amplification: a novel method of long-range molecular haplotyping

Tsai

Pang

et al. 2005

Hum. Mutat.

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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.

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Single-molecule analysis for molecular haplotyping

Cited by 35 publications

References 26 publications

Long-range multilocus haplotype phasing of the MHC

Long-range multilocus haplotype phasing of the MHC

Haplotype Thinking in Lung Disease

Linear allele-specific long-range amplification: a novel method of long-range molecular haplotyping

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