Combinatorial oligonucleotide PCR
AssigneeCapital Genomix
InventorsJill M. Ray, Teri Heiland, Indira Carey
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Abstract
The present invention is directed to a method of detecting gene expression and analysis of both known and unknown genes. In the method utilizing combinatorial oligonucleotide PCR™, no single molecular species of DNA gives rise to more than one fragment in a collection of products which are subsequently amplified and representative of each expressed gene. More specifically, the present invention improves known methods by eliminating the recovery of restriction fragments with two identical ends and furthermore regards amplification and isolation of the restriction fragment utilizing a labeled primer.
Claims70
A method, comprising:
- a) obtaining a DNA attached to an anchor;
- b) cleaving said DNA with a first restriction endonuclease (“A”) to release restriction fragments therefrom;
- c) removing said released fragments while retaining said cleaved DNA using the anchor;
- d) cleaving said retained DNA with a second restriction endonuclease (“B”) different from said first endonuclease, wherein said cleaving results in releasing a fragment mixture comprising:
- i) A/B fragments having nonidentical A and B restriction sites on opposing ends;
- and
- ii) B/B fragments having the same B restriction sites on opposing ends;
- and
- e) separating said A/B fragments from said B/B fragments by a method that comprises:
- i) ligating said fragment mixture with A linkers recognizing and binding said A restriction site and B linkers recognizing and binding said B restriction sites, wherein said A linkers are attached to an anchor, and said B linkers are not attached to such an anchor;
- ii) retaining said A/B fragments by immobilizing fragments using the anchor on the A linkers;
- and
- iii) discarding fragments that have not been immobilized.
Dependencies are inferred from the claim text.