| 摘要 |
A method of amplifying about 10<8>-fold mRNA in a microquantity which is applicable to construction of a cDNA library, subtraction cloning, construction and analysis of a microarray and analysis of gene expression. mRNA in a sample is adsorbed by an oligo(dt)-bonded magnetic bead. Then a double-stranded cDNA is synthesized on the magnetic bead. After attaching a linker having a T7 promoter sequence to the 5'-end, the magnetic bead having the antisense strand cDNA bonded thereto is eliminated. By using the sense strand cDNA in the supernatant as a template, a double-stranded cDNA is synthesized again by using an oligo(dt) primer carrying a linker having an SP6 promoter sequence attached thereto. Then PCR is performed with the use of the known sequences in the linkers at the both ends of this double-stranded cDNA as primers to thereby amplify the cDNA mixture. By using the cDNA mixture, sense strand/antisense strand cRNA is synthesized by T7 and SP6 polymerases.
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