CRACE - A SIMPLE METHOD FOR IDENTIFICATION OF THE 5'-END OF MESSENGER-RNAS
CRACE - A SIMPLE METHOD FOR IDENTIFICATION OF THE 5'-END OF MESSENGER-RNAS
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DOI：
10.1093/nar/23.18.3796
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发表时间：
1995-09-25
影响因子：
14.9
通讯作者：
MARUYAMA, HI
中科院分区：
文献类型：
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作者：
MARUYAMA, IN;RAKOW, TL;MARUYAMA, HI
我们设计了一种识别mRNA 5 '末端的简单方法，其中第一链cDNA通过T4 RNA连接酶环化和/或连接成串联形式，随后将所得单链DNA用作模板，通过聚合酶链式反应（1）（PCR）用基因特异性引物扩增5'末端。该技术已用于秀丽隐杆线虫Unc-13和T12 A2的5 '末端的测定。从cDNA文库中分离的克隆大多缺少5 '端，这是由于逆转录酶使cDNA合成提前终止所致。因此，mRNA的5 ′端定位是分析基因及其启动子的关键步骤。有多种方法可用于定位mRNA末端，包括RNA酶保护、SI定位和引物延伸（2）。这些方法需要相对大量的mRNA，并且通常在鉴定稀有mRNA的5 '端时造成困难。另一种方法被描述为cDNA末端快速扩增（RACE）（3），其中mRNA用基因特异性引物逆转录，cDNA在5 '末端用末端脱氧核苷酸转移酶合成的同源寡核苷酸修饰。加尾cDNA用与尾部互补的寡核苷酸和基因特异性引物进行PCR扩增。通过这种技术，由于加尾反应的效率低下和/或通过均聚引物的非特异性引发，通常难以检测稀有mRNA的5 '端。为了克服这个问题，最近引入了锚定RACE（4-6）。为了防止cDNA的自身连接，该方法需要过量的“锚”寡核苷酸，其连接到第一链cDNA用于随后的PCR扩增。此外，为了防止锚的自连接，必须通过掺入不常见的核苷酸如双脱氧核苷酸来修饰寡核苷酸的3 '末端。修饰的寡核苷酸锚与mRNA的连接需要多步化学和酶促反应以除去5 '端加帽结构。为了简化cDNA和mRNA的多步加工，我们设计了一种方法，我们将其称为cRACE（环状或多联体第一链cDNA介导的RACE）。该方法已用于C.线虫unc-13 mRNA，其编码二酰基甘油/佛波酯受体并在神经传递中起作用（7）。该mRNA仅在一部分神经元中以非常低的水平表达，约为丰富mRNA的0.1%，如肌球蛋白重链，
We have devised a simple method for identification of the 5'end of mRNAs in which the first strand cDNA is circularized and/or joined into a concatemeric form by T4 RNA ligase and the resulting single-stranded DNA is subsequently usedas atemplate for amplification of the 5'end by the polymerase chain reaction (1)(PCR) with gene-specific primers. The technique has been used for determination of the 5'end of Caenorhabditis elegans unc-13 and T12A2. 4 mRNAs.Most clones isolated from cDNA libraries lack their 5'end, due to premature termination of cDNAsynthesis byreverse transcrip-tase. Therefore, 5'-end mapping ofmRNA is acrucial step for the analysis of a gene andits promoter. A variety of methods are available for mapping mRNA ends, including RNase protection, SI mapping and primer extension (2). These methods require relatively large amounts of mRNA and often pose difficulty in identifying the 5'end ofrare mRNAs. An alternative procedure has been described as the rapid amplification of cDNA ends (RACE)(3), in which mRNA is reverse-transcribed with a gene-specific primer and the cDNA is modified at the 5'end with a homo-oligonucleotide synthesized by terminal deoxynucleotidyl transferase. The tailed cDNA is subjected to PCR amplification with a complimentary oligonucleotide to the tail and a gene-specific primer. By this technique, it is often difficult to detect the 5'end of rare mRNAs, due to inefficiency of the tailing reaction and/or non-specific priming by a homopolymeric primer. To overcome this problem, anchored RACE has recently been introduced (4-6). To prevent self ligation of cDNA, the method requires an excess amount of'anchor'oligonucleotide that is linked to first-strand cDNA for subsequent PCR amplification. Furthermore, to prevent self-ligation of the anchors, the 3'end of the oligonucleotide has to be modified by incorporating an unusual nucleotide such as dideoxynucleotides. Ligation of the modified oligonucleotide anchor to mRNA requires multi-step chemical and enzymatic reactions to remove the 5'-end capping structure. To simplify the multi-step processing of cDNA and mRNA, we have devised a method that we have denoted as cRACE (circular or concatemeric first-strand cDNA-mediated RACE). The method has been used for identification of the 5'end of the C. elegans unc-13 mRNA, which encodes a diacylglycerol/phorbol ester receptor and plays a role in neurotransmission (7). The mRNA is expressed in only a subset of neurons at a very low level,-0.1% of abundant mRNAs such as the myosin heavy