Reagents for Preparing Structure-Free DNA and RNA

描述（由申请人提供）：单链ONA和RNA的二级结构是短寡核苷酸探针有效杂交的重要障碍。虽然对给定目标的一些探针可以有效地杂交，但对同一目标的其他探针可能根本不能杂交。使用长度为60个核苷酸的较长的探针是克服这个问题的一种方法。不幸的是，这种探针不能直接用于检测单核苷酸多态性或点突变。使用缺乏二级结构的伪互补（pc） DNA或RNA靶标应该有助于短探针的使用，并能够开发通用寡核苷酸微阵列，其中短探针的每个排列（例如，8-mer）都在阵列上表示。这种阵列非常适合于DNA的重测序，以进行SNP鉴定，并获得用于鉴定目的的遗传谱。我们已经证明，2-氨基腺嘌呤（nATP）和2-硫胸腺嘧啶/2-硫氧嘧啶（sTTP/sUTP）的核苷三磷酸（NTPs）可以被酶结合到DNA或RNA中。这些碱基是伪互补的，因为它们彼此不相互作用，但可以单独与常规碱基互补配对。在这个I期提案中，我们将评估dGTP和dCTP的dNTP类似物是否可以与dnATP和dsTTP一起在引物延伸试验中使用，以产生无结构的单链DNA，该DNA可以高效和特异性地与短DNA或RNA探针杂交。我们自己和其他人的现有数据表明，这一目标应该很容易实现，并将形成评估伪互补DNA和RNA靶标的基础，以便与微阵列格式的短探针结合使用。

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.