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。虽然一些探针与给定的靶标有效杂交，但与相同靶标的其他探针可能根本不杂交。使用长度约为60个核苷酸的较长探针是克服该问题的一种方法。不幸的是，这样的探针不能用于直接检测单核苷酸多态性或点突变。使用缺乏二级结构的假互补（pc）DNA或RNA靶标应便于使用短探针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聚体）在阵列上表示。这样的阵列理想地适合于用于SNP鉴定的DNA重测序和用于鉴定目的的遗传图谱的获取。我们已经表明，核苷三磷酸（NTPs）的2-氨基腺嘌呤（nATP）和2-硫代胸腺嘧啶/2-硫脲（sTTP/sUTP）可以酶促掺入DNA或RNA。这些碱基是伪互补的，因为它们彼此不相互作用，但可以单独配对到常规碱基互补。在本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一起用于引物延伸测定中，以产生可以与短DNA或RNA探针以高效率和特异性杂交的无结构单链DNA。我们和其他人的现有数据表明，这一目标应该是容易实现的，并将形成评估假互补DNA和RNA靶点的基础，用于与短探针结合使用的微阵列格式。

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.