Dataset Information

Digital genotyping and haplotyping with polymerase colonies.

ABSTRACT: Polymerase colony (polony) technology amplifies multiple individual DNA molecules within a thin acrylamide gel attached to a microscope slide. Each DNA molecule included in the reaction produces an immobilized colony of double-stranded DNA. We genotype these polonies by performing single base extensions with dye-labeled nucleotides, and we demonstrate the accurate quantitation of two allelic variants. We also show that polony technology can determine the phase, or haplotype, of two single- nucleotide polymorphisms (SNPs) by coamplifying distally located targets on a single chromosomal fragment. We correctly determine the genotype and phase of three different pairs of SNPs. In one case, the distance between the two SNPs is 45 kb, the largest distance achieved to date without separating the chromosomes by cloning or somatic cell fusion. The results indicate that polony genotyping and haplotyping may play an important role in understanding the structure of genetic variation.

SUBMITTER: Mitra RD

PROVIDER: S-EPMC156303 | biostudies-literature | 2003 May

REPOSITORIES: biostudies-literature

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Publications

Digital genotyping and haplotyping with polymerase colonies.

Mitra Robi D RD Butty Vincent L VL Shendure Jay J Williams Benjamin R BR Housman David E DE Church George M GM

Proceedings of the National Academy of Sciences of the United States of America 20030502 10

Polymerase colony (polony) technology amplifies multiple individual DNA molecules within a thin acrylamide gel attached to a microscope slide. Each DNA molecule included in the reaction produces an immobilized colony of double-stranded DNA. We genotype these polonies by performing single base extensions with dye-labeled nucleotides, and we demonstrate the accurate quantitation of two allelic variants. We also show that polony technology can determine the phase, or haplotype, of two single- nucle ...[more]

PMID: 12730373

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Project description:Background: Human neutrophil antigen-3A (HNA-3A) and human neutrophil antigen-3B (HNA-3B) are generated by a single-nucleotide polymorphism (rs2288904, c.461G > A) in exon 7 of the choline transporter-like protein-2 gene (CTL2, also known as SLC44A2). Antibodies to HNA-3 can be generated following blood transfusion or other factors resulting in exposure to HNA-3 antigens. These antibodies can cause transfusion-related acute lung injury (TRALI) or neonatal alloimmune neutropenia (NAIN). This study describes a sensitive and specific TaqMan real-time polymerase chain reaction (PCR) method to screen for the HNA-3 genotype using specific primers and probes designed to detect allelic polymorphisms. Considering the high sensitivity and accuracy of droplet digital PCR (ddPCR) in the identification of the rare SLC44A2*2 allele, we used this technique to identify blood donors with the rare HNA-3B antigen and calculate the allele frequency of SLC44A2 in mixed populations with different proportions. Methods: DNA samples purified from 208 donors in northwest China were subjected to TaqMan real-time PCR to detect allelic polymorphisms in SLC44A2. The results were confirmed by Sanger sequencing. The rare HNA-3B antigen was detected by ddPCR. SLC44A2 frequency was determined by two-channel ddPCR. Results: The genotypes of all DNA samples were detected by the TaqMan real-time PCR using specific probes for HNA-3, and the results were consistent with the Sanger sequencing results in respect to the HNA-3A and HNA-3B polymorphisms. The allele frequencies of SLC44A2*1 and SLC44A2*2 in the 208 donors in northwest China were 64.9% (95% confidence interval [CI], 59%-70.8%) and 35.1% (95% CI, 29.2%-41%), respectively. The ratio of SLC44A2*2 alleles was accurately detected in all blood pools by ddPCR but not by TaqMan real-time PCR. This allowed for the SLC44A2 frequency in the population to be accurately inferred. Conclusion: This new method of detecting SLC44A2 alleles was highly sensitive and specific, as confirmed by Sanger sequencing. ddPCR using the designed probes resulted in successful detection of the rare HNA-3B antigen. Furthermore, we successfully detected the rare HNA-3B antigen and inferred the SLC44A2 frequency by ddPCR using the probes that we designed.

Dataset Information

Digital genotyping and haplotyping with polymerase colonies.

Publications

Digital genotyping and haplotyping with polymerase colonies.

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