Heteropolymeric Triplex-Based Genomic Assay® to Detect Pathogens or Single-Nucleotide Polymorphisms in Human Genomic Samples

Jasmine I Daksis,Glen H Erikson,Cecile Fairhead

doi:10.1371/journal.pone.0000305

Abstract

Human genomic samples are complex and are considered difficult to assay directly without denaturation or PCR amplification. We report the use of a base-specific heteropolymeric triplex, formed by native duplex genomic target and an oligonucleotide third strand probe, to assay for low copy pathogen genomes present in a sample also containing human genomic duplex DNA, or to assay human genomic duplex DNA for Single Nucleotide Polymorphisms (SNP), without PCR amplification. Wild-type and mutant probes are used to identify triplexes containing FVL G1691A, MTHFR C677T and CFTR mutations. The specific triplex structure forms rapidly at room temperature in solution and may be detected without a separation step. YOYO-1, a fluorescent bis-intercalator, promotes and signals the formation of the specific triplex. Genomic duplexes may be assayed homogeneously with single base pair resolution. The specific triple-stranded structures of the assay may approximate homologous recombination intermediates, which various models suggest may form in either the major or minor groove of the duplex. The bases of the stable duplex target are rendered specifically reactive to the bases of the probe because of the activity of intercalated YOYO-1, which is known to decondense duplex locally 1.3 fold. This may approximate the local decondensation effected by recombination proteins such as RecA in vivo. Our assay, while involving triplex formation, is sui generis, as it is not homopurine sequence-dependent, as are “canonical triplexes”. Rather, the base pair-specific heteropolymeric triplex of the assay is conformation-dependent. The highly sensitive diagnostic assay we present allows for the direct detection of base sequence in genomic duplex samples, including those containing human genomic duplex DNA, thereby bypassing the inherent problems and cost associated with conventional PCR based diagnostic assays.

Highlights

Triplex nucleic acids have attracted attention due to their potential regulation of DNA transcription, replication, recombination, chromosome packing, and potential applications in gene therapy [1,2,3,4,5,6,7,8]
We demonstrate the Genomic AssayH, carried out homogeneously in bulk solution, whereby Single Nucleotide Polymorphisms (SNP) or pathogens in samples containing human genomic double-stranded DNA (dsDNA) are directly detected by fluorescent signal
CFTR 2789+5GRA-WT25C or mutant probe CFTR 2789+5GRA-MUT25C (Supplemental Table S5). These results collectively demonstrate the high level of efficiency and specificity of the Genomic AssayH when practised in bulk solution with 25-mer ssDNA probes and YOYO-1, to directly detect mismatches between probes and non-denatured, non-amplified human genomic dsDNA targets, over a broad range of genomic target concentrations

Summary

Introduction

Triplex nucleic acids have attracted attention due to their potential regulation of DNA transcription, replication, recombination, chromosome packing, and potential applications in gene therapy [1,2,3,4,5,6,7,8]. Substantial deviations from strict binding rules are understood to preclude triplex formation [9,10,11,12,13] The utility of such ‘‘canonical triplexes’’ is severely limited due to the requirement of long uninterrupted homopurine sequences in the double-stranded DNA (dsDNA). Specific heteropolymeric triplex formation between non-denatured dsDNA and a ssDNA of mixed base sequence can occur without strand displacement, duplex invasion or the action of recombination proteins, such as recA This triplex binding capability was used to demonstrate that the Triplex AssayH can accurately detect 1-, 2-, or 3-bp mutations, deletions or insertions in synthetic dsDNA targets and in homozygous or heterozygous polymerase chain reaction (PCR) amplified dsDNA amplicons of varied GC content [14]

Methods

Results

Conclusion