A Lishanski1. 1. Advanced Diagnostics Group, Dade Behring Inc, San Jose, CA 95135, USA. alla_lishanski@dadebehring.com
Abstract
BACKGROUND: New methods are required for the exploration of the human genome by discovering sequence variations. This study evaluated the performance of a new method for screening a large number of samples for several DNA polymorphisms. METHODS: We used a homogeneous method based on inhibition of spontaneous branch migration by any sequence difference between two molecules of PCR-amplified DNA. A set of four PCR primers is required: a forward primer, either biotinylated or labeled with digoxigenin, and two reverse primers that share a priming domain but have different "tail" sequences at their 5' ends. After PCR amplification, denaturation and reannealing of the single DNA strands produce doubly labeled cruciform structures, which dissociate by strand exchange. The presence of two different alleles in a sample causes complete inhibition of dissociation, and the association of biotin and digoxigenin is homogeneously detected using luminescent oxygen channeling immunoassay. RESULTS: The 90 samples of the Human Variation Panel (Coriell Cell Repositories) were screened for nine known single-nucleotide polymorphisms (SNPs) and one 5-bp deletion. The average signal-to-background ratio varied from approximately 10 to 20. The frequency of the predominant allele for different SNPs varied from 51% to 88% overall. For some SNPs, it varied among the nine ethnic groups, e.g., 25-85% (average, 51%) for one SNP. The average heterozygosity varied from 0.17 to 0.54 and as much as 0.2-0. 9 (average, 0.54) for one of the SNPs. CONCLUSION: The method allows simple and rapid screening of a large number of samples for the presence of multiple alleles.
BACKGROUND: New methods are required for the exploration of the human genome by discovering sequence variations. This study evaluated the performance of a new method for screening a large number of samples for several DNA polymorphisms. METHODS: We used a homogeneous method based on inhibition of spontaneous branch migration by any sequence difference between two molecules of PCR-amplified DNA. A set of four PCR primers is required: a forward primer, either biotinylated or labeled with digoxigenin, and two reverse primers that share a priming domain but have different "tail" sequences at their 5' ends. After PCR amplification, denaturation and reannealing of the single DNA strands produce doubly labeled cruciform structures, which dissociate by strand exchange. The presence of two different alleles in a sample causes complete inhibition of dissociation, and the association of biotin and digoxigenin is homogeneously detected using luminescent oxygen channeling immunoassay. RESULTS: The 90 samples of the Human Variation Panel (Coriell Cell Repositories) were screened for nine known single-nucleotide polymorphisms (SNPs) and one 5-bp deletion. The average signal-to-background ratio varied from approximately 10 to 20. The frequency of the predominant allele for different SNPs varied from 51% to 88% overall. For some SNPs, it varied among the nine ethnic groups, e.g., 25-85% (average, 51%) for one SNP. The average heterozygosity varied from 0.17 to 0.54 and as much as 0.2-0. 9 (average, 0.54) for one of the SNPs. CONCLUSION: The method allows simple and rapid screening of a large number of samples for the presence of multiple alleles.