ENHANCED DIRECT AMPLIFICATION OF GUTHRIE CARD DNA FOLLOWING SELECTIVE ELUTION OF PCR INHIBITORS
ENHANCED DIRECT AMPLIFICATION OF GUTHRIE CARD DNA FOLLOWING SELECTIVE ELUTION OF PCR INHIBITORS
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DOI：
10.1093/nar/23.18.3788
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发表时间：
1995-09-25
影响因子：
14.9
通讯作者：
HOPFER, SM
中科院分区：
文献类型：
--
作者：
MAKOWSKI, GS;DAVIS, EL;HOPFER, SM
古特里卡片DNA代表了一个重要但相对未充分利用的基因组材料库，用于遗传筛选（1）。这个问题是由于血斑中捕获的DNA量有限以及全血PCR抑制剂的存在造成的（2-4）。在本文中，我们展示了一种简单的方法，以提高扩增效率的古特里卡DNA的PCR污染物的选择性洗脱，同时保留DNA在可分离的形式。之前，我们发现囊膜纤维化（CF）AF 508突变的PCR可以直接在古特里卡片标本上进行，以获得小尺寸扩增子（98 bp）（5）。然而，较大CF基因组区域（491 bp）的扩增是不可再现的（6）。扩增不一致的原因尚不清楚，但可能是由于高水平的血斑成分释放到PCR反应混合物中所致（图1A）。随后通过SDS-PAGE的分析揭示了两个主要电泳条带的存在（图1B）。基于它们的相对丰度、与红细胞（rbc）溶血产物的共迁移和蛋白质印迹（未显示），这些蛋白质被暂时鉴定为rbc碳酸酐酶（30 kDa，与免疫球蛋白轻链共迁移）和珠蛋白（14 kDa）。为了从Guthriecards中去除全血污染物，我们尝试了许多DNA纯化方案，包括用SDS、蛋白酶K消化和苯酚：氯仿提取处理。我们发现这些导致DNA和PCR抑制剂同时释放的程序是复杂的、耗时的和不可靠的（未显示）。相反，血斑材料（3 mm直径穿孔器的1/4）在1 ml水中的预温育（两次，30分钟）导致释放到PCR反应混合物中的几乎所有血斑污染物的消除（图1A和B）。PCR后反应混合物的SDS-PAGE分析显示，从未洗涤的样品释放的蛋白质形成大的不溶性高分子量复合物（即变性聚集体）几乎没有进入堆积凝胶（闭合三角形）或浓缩在分离凝胶中（空心三角形）尽管未洗涤的（-）滤纸的扩增导致98 bp扩增子的可观察产物，溴化乙锭染色条带的总强度通常较低（图2A）。当以单一（491 bp）或双链体（425/491 bp）PCR形式扩增较大CF基因组区域时，明显信号的缺乏被放大（图2B和C）。与此相反，PCR扩增洗涤后的（+）古特里卡标本，
Guthrie card DNA represents an important yet relatively underutilized repository of genomic material for genetic screen-ing (1). This problem results from the limited amount of DNA entrapped within bloodspots and the presence of whole blood PCR inhibitors (2-4). In this paper we demonstrate a simple method to enhance the amplification efficiency of Guthrie card DNA by the selective elution of PCR contaminants while retaining DNA in amplifiable form. Previously we found that PCR for thecystic fibrosis (CF) AF508 mutation could be performed directly on Guthrie card specimens for small size amplicons (98 bp)(5). Amplification of larger CF genomic regions (491 bp) was not, however, reproduc-ible (6). The reason for inconsistentamplification is unclear, but may result from the release ofhigh levels of bloodspot components into the PCR reaction mix (Fig. IA). Subsequent analysis by SDS-PAGE revealed the presence of two major electrophoretic bands (Fig. IB). Based on their relative abundance, comigation with red blood cell (rbc) hemolysate and Western blotting (not shown) these proteins were tentatively identified as rbc carbonic anhydrase (30 kDa, comigrating with immunoglobulin light chains) and globin (14 kDa). To remove whole blood contaminants from Guthriecards, we attempted a number of DNA purification schemes including treatment with SDS, proteinase K digestion and phenol: chloroform extraction. We found these procedures, which result in the simultaneous release of DNA as well as PCR inhibitors, to be complex, time-consuming and unreliable (not shown). In contrast, pre-incubation of bloodspot material (1/4 of a 3 mm diameter punch) in 1 ml water (twice, 30 min) resulted in elimination of virtually all bloodspot contaminants released into the PCR reaction mix (Fig. IA and B). SDS-PAGE analysis of the post-PCR reaction mix revealed that protein released from unwashed specimens formed large insoluble high molecular weight complexes (ie denatured aggregates) that barely entered the stacking gel (closed triangle) or concentrated at theresolving gel (open triangle)(Fig. IB).Although amplificationof unwashed (-) filter paper resulted in observable product for 98 bp amplicons, overall intensity of the ethidium bromide-stained bands was generally lower (Fig. 2A). The lack of appreciable signal was magnified when larger CF genomic regions were amplified in single (491 bp) or duplex (425/491 bp) PCR format (Fig. 2B and C). In contrast, PCR amplification ofwashed (+) Guthrie card specimens resulted in