RAPID PRODUCTION OF FULL-LENGTH CDNAS FROM RARE TRANSCRIPTS - AMPLIFICATION USING A SINGLE GENE-SPECIFIC OLIGONUCLEOTIDE PRIMER
RAPID PRODUCTION OF FULL-LENGTH CDNAS FROM RARE TRANSCRIPTS - AMPLIFICATION USING A SINGLE GENE-SPECIFIC OLIGONUCLEOTIDE PRIMER
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DOI：
10.1073/pnas.85.23.8998
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发表时间：
1988-12-01
影响因子：
11.1
通讯作者：
MARTIN, GR
中科院分区：
文献类型：
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作者：
FROHMAN, MA;DUSH, MK;MARTIN, GR
我们设计了一种简单而有效的cDNA克隆策略，克服了在获得低丰度mRNAs全长cDNA克隆时遇到的许多困难。本质上，cDNA是通过使用DNA聚合酶链式反应技术来扩增转录本中单个点与3‘’或5‘端之间区域的拷贝而产生的。这种扩增所需的最小信息是要克隆的信使核糖核酸中的一小段序列。由于cDNA可以在一天内产生，第二天用Southern blotting检测，并且很容易被克隆，因此可以快速地产生大量稀有转录本的全长cDNA克隆。此外，凝胶电泳法分离扩增的cDNA允许在克隆前根据大小进行精确选择，从而有助于分离代表不同mRNAs的cDNA，例如通过选择性剪接或使用替代启动子产生的那些。通过从INT-2基因中分离出表达四种不同转录本的小鼠基因Int-2，证明了该方法的有效性，其中最长的转录本是.apprxeq。2.9千碱基。经过0.05%的筛选，共分离到29个独立的INT-2克隆。序列分析表明，所有4个int-2mRNAs的3‘’和5‘’末端都被这些克隆准确地表达出来。
We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3'' or 5'' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is .apprxeq. 2.9 kilobases. After < 0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3'' and 5'' ends of all four int-2 mRNAs were accurately represented by these clones.