SITE SPECIFIC CHEMICAL METHODS FOR CLEAVING GENOMIC DNA

染色体的物理图谱将通过以下方法得到促进 将大的DNA分解成可处理的片段或在 感兴趣的遗传标记。我们的长期目标是提供 在巨型数据库中有效切割单个位置的化学方法和 Gigabase DNA(人类染色体)。寡核苷酸导向的三螺旋结构 构型是一种通用的序列特异性识别方法 15个碱基对的DNA。由于识别位置的长度，在 正式意义上，这是比那更具体序列的一百万倍 可与大多数限制酶一起使用。重要的是要确定 这种切割特异性的全部潜力是否能够实现。 我们的计划结合了合成有机化学、生物物理方法和 分子生物学研究结构与功能的关系 在制造用于识别和修饰DNA的试剂方面 生物聚合物。具体目标是：(1)检测高产双链 5‘-(Pur)/m(PYR)/n-3’靶序列在兆数据库中的切割反应 利用新型的嘧啶寡核苷酸合成DNA 3‘端的N-溴乙酰胺，(2)测试嘌呤-N-溴乙酰胺- 寡核苷酸用于单位点的高产量双链切割 百万碱基DNA和检测DNA在内部GC碱基对的切割 利用一种新设计的碱(雾草碱-N-溴乙酰胺)，(3)研究 序列合成效应、修饰碱基和错配 通过协同结合寡核苷酸在DNA中的位置，(4)研究 含新型D/3碱基的寡核苷酸的热力学和实验 它们用于靶向三核苷酸重复序列，如(CAG)n，(5) 表征在RecA-中形成的联合分子中的中间体- 用亲和力切割方法在RecA上介导链交换 核蛋白细丝。

Physical mapping of chromosomes would be facilitated by methods of breaking large DNA into manageable fragments or cutting uniquely at genetic markers of interest. Our long-term objective is to provide chemical methods for efficient cleavage at single sites in megabase and gigabase DNA (human chromosomes). Oligonucleotide-directed triple helix formation is a versatile method for the sequence specific recognition of 15 base pairs of DNA. Due to the length of the recognition site, in a formal sense, this is one million times more sequence specific than that available with most restriction enzymes. It is important to determine whether the full potential of this cleavage specificity can be realized. Our program combines synthetic organic chemistry, biophysical methods, and molecular biology to learn about the relationship of structure to function in the area of creating reagents for recognition and modification of DNA biopolymers. Specific aims are (1) test the high yield double strand cleavage reactions at 5'-(pur)/m.(pyr)/n-3' target sequences in megabase DNA utilizing a new class of pyrimidine oligonucleotides equipped with the N-bromoacetamide at the 3' end, (2) test purine N-bromoacetamide- oligonucleotides for high yield double strand cleavage of single sites in megabase DNA and examine cleavage of DNA at an internal GC base pair utilizing a novel designed base (nebularine-N-bromoacetamide), (3) study sequence composition effects, modified bases, and mismatches at a single site in DNA by cooperatively binding oligonucleotides, (4) study the thermodynamics of oligonucleotides containing the novel base D/3 and test their use for targeting trinucleotide repeats such as (CAG)n, (5) characterize the intermediates in the joint molecule formed in a RecA- mediated strand exchange using affinity cleavage methods on a RecA nucleoprotein filament.