Optimization of long-distance PCR using a transposon-based model system.
Optimization of long-distance PCR using a transposon-based model system.
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使用基于转座子的模型系统优化长距离 PCR。
DOI：
10.1101/gr.2.1.51
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
E. Rose
中科院分区：
文献类型：
--
作者：
L. Ohler;E. Rose
无论靶模板序列或结构如何，以高特异性和保真度扩增常规长PCR产物（5-25 kb）的能力将为基因组作图和测序努力提供显著益处。尽管偶尔有报道描述了长PCR产物的产生，但这样的结果难以复制，并且经常利用探针杂交来从非特异性扩增的DNA中鉴定特异性产物。特异性PCR产物的产生通常限于小于3 kb的靶模板。为了扩展标准PCR扩增的有效范围，可能需要利用替代的反应条件和/或组分，例如新型热稳定DNA聚合酶或辅助蛋白。我们描述了使用一个模型系统，以系统地评估方法的变化，可能使有效的远程PCR。具体地，转座子Tn 5supF已被用于在携带相同插入物的噬菌体克隆的单独分离物内引入随机相同的已知引物结合位点。基于转座子的PCR允许我们仅使用一组引物来研究大小和序列不同的DNA片段的扩增。在目前的研究中，我们描述的条件，使PCR扩增的特定DNA模板的大小范围高达9 kb。我们的方法的一些关键特征包括使用重组嗜热栖热菌（rTth）DNA聚合酶，添加明胶的反应混合物，使用蜡介导的“热启动”，最后，使用自动段延伸热循环。这些结果还提供了其他方法的见解，这些方法可能进一步提高我们进行长距离PCR的能力。（250字处删节）
The ability to amplify routinely long PCR products (5-25 kb) with high specificity and fidelity, regardless of target template sequence or structure, would provide significant benefits to genome mapping and sequencing endeavors. Although occasional reports have described the generation of long PCR products, such results have been difficult to replicate and have frequently utilized probe hybridization to identify the specific product from nonspecific amplified DNA. Production of specific PCR products has generally been limited to target templates of less than 3 kb. To extend the effective range of standard PCR amplification, it may be necessary to utilize alternative reaction conditions and/or components, such as novel thermostable DNA polymerases or accessory proteins. We describe the use of a model system to evaluate systematically methodological changes that might enable efficient long-range PCR. Specifically, the transposon Tn5supF has been used to introduce randomly identical, known primer binding sites within separate isolates of phage clones carrying identical inserts. Transposon-based PCR allows us to study amplification of DNA fragments that vary in size and sequence using only a single set of primers. In the present studies, we describe conditions that enable PCR amplification of specific DNA templates ranging in size up to 9 kb. Some of the key features of our methodology include the use of recombinant Thermus thermophilus (rTth) DNA polymerase, the addition of gelatin to the reaction mixture, the use of wax-mediated "hot starts" and, lastly, the use of auto-segment extension thermocycling. These results also provide insights into additional approaches that might further enhance our ability to perform long-distance PCR.(ABSTRACT TRUNCATED AT 250 WORDS)