PloS ONE：三链检测法分析双链基因组DNA
直接检测双链核酸中的碱基序列一直是一个无法解决的难题。在3月21日的国际知名网上开放杂志PloS ONE上，Ingeneus Research公司会发表一篇名为“杂聚三链基因组检测方法分析人类基因组样本中的病原或单核苷酸多态性”（Heteropolymeric Triplex-Based Genomic Assay to Detect Pathogens or Single-Nucleotide Polymorphisms in Human Genomic Samples）的文章。
在文章中，研究人员给出了从样本得到的病原分析数据，以及人类基因组双链DNA和SNP分析结果。这些分析能够平行进行，并且可能在室温下进行。在5分钟后，研究人员能够监控反应情况。这种高灵敏诊断分析能够实现对人类基因组双链样本中碱基序列的直接检测，无需再使用PCR方法，从而避免了这种方法的内在问题并费用。
Ingeneus Research的首席科学家Jasmine Daksis表示，他们已经逐渐研制出了杂聚三链分析（heteropolymeric triplex）。研究人员首先从合成的50-mer双链靶标开始，开发出了能够分析人类基因组样本的方法。这项分析利用YOYO-1(一种bisintercalator)来解压缩双链靶标，这样使双链核酸能更容易与oligo ssDNA探针反应。双链中任何序列都能被特异性分析。研究人员推测特定的三链结合创造出另外的凹槽供另外的YOYO-1分子插入。
该公司的研究团队决定不将注意力集中在改善探针化学性质方面，转而开发一种流体注射设备。这种叫做Genome Flow的设备使用了FIALab装置的硬件。研究人员希望能够尽快发表利用Genome Flow设备进行基因组样本病原或SNP的三链分析。
部分英文原文：
PloS ONE，February 16, 2007; Accepted: February 25, 2007; Published: March 21, 2007
Heteropolymeric Triplex-Based Genomic Assay® to Detect Pathogens or Single-Nucleotide Polymorphisms in Human Genomic Samples
Ingeneus Research, Mississauga, Ontario, Canada
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.
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