Format

Send to

Choose Destination
PLoS One. 2015 Mar 4;10(3):e0118270. doi: 10.1371/journal.pone.0118270. eCollection 2015.

A rapid molecular approach for chromosomal phasing.

Author information

1
Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, United States of America.
2
Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America; Program in Medical and Population Genetics and Stanley Center for Psychiatric Research, Cambridge, Massachusetts, United States of America.
3
Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.

Abstract

Determining the chromosomal phase of pairs of sequence variants - the arrangement of specific alleles as haplotypes - is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb) without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well. Physically linked alleles partition into the same droplets, revealing their chromosomal phase in the co-distribution of fluorophores across droplets. We demonstrated the accuracy of this method by phasing members of trios (revealing 100% concordance with inheritance information), and demonstrate a common clinical application by phasing CFTR alleles at genomic distances of 11-116 kb in the genomes of cystic fibrosis patients. Drop-Phase is rapid (requiring less than 4 hours), scalable (to hundreds of samples), and effective at long genomic distances (200 kb).

PMID:
25739099
PMCID:
PMC4349636
DOI:
10.1371/journal.pone.0118270
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Public Library of Science Icon for PubMed Central
Loading ...
Support Center