SUBTRACTION METHOD OF DOUBLE-STRANDED cRNA DERIVED FROM TRIMMED cDNA

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