Patent ID: 7919238

Claim:
A method of identifying an alternatively spliced RNA molecule in conjunction with a normally spliced counterpart RNA molecule, comprising the steps of: (a) obtaining a first population of cDNA molecules from a biological sample representing a first physiological condition and a second population of cDNA molecules from a biological sample representing a second physiological condition; (b) attaching a first selectable tag to cDNA molecules of the first cDNA population and a second selectable tag to cDNA molecules of the second cDNA population, wherein the first and second selectable tags are different; (c) combining, denaturing and annealing cDNA molecules from both the first and second cDNA populations, to obtain a mixed population of cDNA molecules including cross-hybridized double-stranded cDNA that comprises both a single strand that is from the first population of cDNA and that is attached to the first selectable tag, and a single strand that is from the second population of cDNA and that is attached to the second selectable tag; (d) isolating cross-hybridized double-stranded cDNA that comprises both the first and second selectable tags from the mixed population; (e) selecting from the cross-hybridized double-stranded cDNA isolated in step (d), cross-hybridized double-stranded cDNA which comprises at least one area of mismatched sequence present as an unhybridized single-stranded nucleic acid; (f) coupling at one end of the 3′ end of one strand of a cross-hybridized double-stranded cDNA molecule selected in step (e) to the 5′ end of the other strand of said cross-hybridized double-stranded cDNA molecule to form a loop and produce a coupled cross-hybridized double-stranded cDNA molecule that comprises at least one area of mismatched sequence, wherein when denatured said coupled cross-hybridized double-stranded cDNA molecule is a single linear single stranded nucleic acid molecule; (g) denaturing the coupled cross-hybridized double-stranded cDNA molecule to form a single linear single stranded nucleic acid molecule; and (h) analyzing the sequence of the linear single stranded nucleic acid molecule derived from the cross-hybridized double stranded cDNA to identify the sequence of an alternatively spliced RNA molecule through comparison of sequences derived from the first population of cDNA to sequences derived from the second population of cDNA.