Nucleic Acids Research, 1995, Vol. 23, No. 18 3685-3690
© 1995
METHODS |
Description of the entire mRNA population by a 3' end cDNA fragment generated by class IIS restriction enzymes
Okayama Cell Switching Project, ERATO, JRDC 103-5 Tanakamonzencho, Sakyo-ku, Kyoto 606, Japan
Received June 16, 1995. Revised August 17, 1995. Accepted August 17, 1995.
A novel means of recording the expression status of the total gene population is described. Digestion of cDNA by class IIS restriction enzymes produces a fragment with a poly (A) stretch and a 5' overhang with an unknown sequence. This fragment contains information such as the class IIS enzyme that cuts cDNA nearest to the poly (A) stretch, the sequence of the 5' overhang, and the size of the fragment. Expressed genes can be discriminated and displayed by the fragment as follows: (i) cut the cDNA with one class IIS restriction enzyme; (ii) ligate the digested cDNA to that from a pool of 64 biotinylated adaptors cohesive to all possible overhangs; (iii) digest by other two class IIS enzymes; (iv) recover the ligated molecules with streptavidin-coated paramagnetic beads; (v) perform PCR with the adaptor-primer and an anchored oligo-dT primer (vi) separate the amplified fragments by denaturing polyacrylamide gel electrophoresis. Repeat the experiment with 64 adaptors, three enzymes and three anchored oligo-dT primers displays most of the expressed genes. Because redundancy is minimized, this technique is also ideal for generating tags for expressed genes, with which to construct a transcript map of the genome.
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