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KMID : 0370219870310020116
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Yakhak Hoeji
1987 Volume.31 No. 2 p.116 ~ p.125
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Rapid and Efficient Molecular Cloning of Rat Liver Full-length LDH A-cDNA
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노옥경/Rho OK
배석철/이승기/Bae SC/Lee SK
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Abstract
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It is still difficult and time consuming to obtain cDNA sequences that contain the entire nucleotide sequence of the corresponding mRNA. A rapid and high efficient cloning method to obtain full-length cDNA segments is thus developed. The cloning procedure described here consists of the construction of oligo(dT)-tailed vector primer using pWR34 plasmid, polyadenylation of mRNA-cDNA heteroduplex using terminal deoxytransferase, and replacement of mRNA strand with DNA by RNase H and DNA polymerase I. The restriction endonuclease analysis shows that the size of inserted-cDNA is in the range of 1.5-4.0kb long suggesting that most of cloned cDNA are full-length or nearly full-length cDNA. The plasmid-DNA recombinants obtained were 4 X 105-106 per mcg of rat liver poly (A+)mRNA, which is 4 to 10 fold higher cloning efficiency in comparison to the presently used methods for full-length cDNA cloning. The results indicate that the described cloning system is much simpler, less time consuming, and very efficient cloning method to construct a cDNA library.
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KEYWORD
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