| 摘要 |
<P>PROBLEM TO BE SOLVED: To provide a subtraction method capable of efficiently conducting subtraction, by using mRNAs derived from a tester in a very small amount, and capable of cloning a gene of which the expression level is fluctuated under a specified condition. <P>SOLUTION: This subtraction method comprises dividing all cDNAs derived from the mixed mRNAs into three equal parts, fragmenting the cDNAs of the divided three fractions with three kinds of restriction enzymes, respectively, mixing again the fragmented cDNAs of the three fractions, trimming the cDNAs to have 2,000 base pairs (bp) or less (1,000 pb in average) counted from the 3'-terminal, coupling the cDNAs with linkers, and amplifying the cDNAs by a PCR technique, so as to synthesize the cDNAs. Further, the substraction is conducted by synthesizing sense and antisense strand biotin-labeled double-stranded cRNAs by using the synthesized cDNAs as templates, using the double-stranded cRNAs as drivers to form hybrids together with the cDNAs derived from the tester, and removing the biotinylated hybrids by using an immobilized streptavidin. A substraction library is structured by inserting the remained cDNA into a plasmid. <P>COPYRIGHT: (C)2005,JPO&NCIPI |
