Simple cDNA normalization using kamchatka crab duplex-specific nuclease
Simple cDNA normalization using kamchatka crab duplex-specific nuclease
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DOI：
10.1093/nar/gnh031
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发表时间：
2004-02-01
影响因子：
14.9
通讯作者：
Shagin, DA
中科院分区：
文献类型：
--
作者：
Zhulidov, PA;Bogdanova, EA;Shagin, DA
我们开发了一种新的简单的cDNA归一化方法[称为双链特异核酸酶(DSN)归一化]，该方法可以有效地用于富含全长cDNA序列的样品。DSN的归一化包括DNA的变性-再结合，由丰富的转录本形成的双链(DS)片段的降解，以及均衡的单链(Ss)DNA片段的PCR扩增。这种方法的关键是降解在使用堪察加螃蟹DSN重新结合时形成的DS组分，正如最近所描述的那样。与双链DNA和RNA相比，这种耐热的酶在DNA-RNA杂化双链中表现出强烈的切割双链DNA和DNA的偏好，与序列长度无关。我们制定了第一链cDNA[当有Poly(A)(+)RNA可用时]和扩增的cDNA(当只能获得总RNA时)的标准化方案。这两种方案都在模型实验中使用人类骨骼肌基因进行了评估。为了进一步说明DSN归一化技术的有效性，我们还利用DSN归一化技术对海洋软体动物海兔(Aplysia Calfornica)的神经组织进行了归一化处理。
We developed a novel simple cDNA normalization method [termed duplex-specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full-length cDNA sequences. DSN normalization involves the denaturation-reassociation of cDNA, degradation of the double-stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single-stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA-RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first-strand cDNA [when poly(A)(+) RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique.