Reagents for Preparing Structure-Free DNA and RNA

描述(申请人提供)：单链ONA和RNA的二级结构是短链寡核苷酸探针有效杂交的重要障碍。虽然针对给定目标的一些探针可以高效杂交，但针对同一目标的其他探针可能根本不会杂交。使用长度约为60个核苷酸的较长探针是克服这一问题的一种方法。不幸的是，这样的探针不能直接用于检测单个核苷酸的多态性或点突变。使用缺乏二级结构的伪互补(PC)DNA或RNA靶标应促进短探针的使用，并使通用寡核苷酸微阵列的开发成为可能，其中短探针(例如，8-聚体)的每个排列都表示在阵列上。这种阵列非常适合用于SNP鉴定的DNA重测序和用于鉴定目的的基因图谱的获取。我们已经证明了2-氨基腺嘌呤(NATP)和2-硫代胸腺嘧啶/2-硫尿嘧啶(STTP/sUTP)的核苷三磷酸盐(NTPs)可以被酶结合到DNA或RNA中。这些碱基是假互补的，因为它们彼此不相互作用，但可以单独与规则的碱基互补配对。在这个第一阶段的提案中，我们将评估dNTP类似物dGTP和dCTP是否可以与dnATP和dsTTP一起用于引物延伸分析，以产生能够与短DNA或RNA探针高效和特异杂交的无结构单链DNA。来自我们和其他人的现有数据表明，这一目标应该很容易实现，并将成为评估与微阵列形式的短探针结合使用的伪互补DNA和RNA靶标的基础。

DESCRIPTION (provided by applicant): Secondary structure in single-stranded ONA and RNA is a significant barrier to the efficient hybridization of short oligonucleotide probes. While some probes to a given target hybridize efficiently, other probes to the same target may not hybridize at all. Use of longer probes, on the order of 60 nucleotides in length, is one way to overcome this problem. Unfortunately, such probes cannot be used to directly detect single nucieotide polymorphisms or point mutations. Use of pseudo-complementary (pc) DNA or RNA targets that lack secondary structure should facilitate the use of short probes and enable the development of generic oligonucleotide microarrays wherein every permutation of a short probe (e.g., an 8-mer) is represented on the array. Such arrays are ideally suited to the resequencing of DNA for SNP identification and to the acquisition of genetic profiles for identification purposes. We have shown that nucleoside triphosphates (NTPs) of 2-aminoadenine (nATP) and 2-thiothymine/2-thiouracii (sTTP/sUTP) can be enzymatically incorporated into DNA or RNA. These bases are pseudo-complementary since they don't interact with each other but can individually pair to the regular base complement. In this Phase I proposal we will evaluate whether dNTP analogues of dGTP and dCTP can be used together with dnATP and dsTTP in a primer extension assay to generate structure-free single-stranded DNA that can hybridize to short DNA or RNA probes with high efficiency and specificity. Existing data from ourselves and others indicate that this goal should be readily attainable and would form the basis for evaluating pseudo-complementary DNA and RNA targets for use in conjunction with short probes in a microarray format.