Method for identification, separation and quantitative measurement of nucleic acid fragments

A method for detecting polymorphism comprising:

• (a) reverse transcribing an RNA population to provide a double-stranded cDNA population;
• (b) digesting said cDNA population with one or more restriction endonucleases having a degenerate recognition or cleavage sequence, wherein the degenerate recognition or cleavage sequence is represented by the formula of Nm, where N is the extent of degeneracy, m is the number of degenerate bases, and m is 1-5, to produce restriction fragments having Nmdifferent single-stranded overhangs for each restriction endonuclease;
• (c) ligating said restriction fragments to a series of adapters lacking restriction endonuclease sites, each adapter having a sequence complementary to one of said overhangs such that restriction fragments having identical overhangs are ligated to the same adapter;
• (d) amplifying said restriction fragments for no more than 25 cycles;
• (e) sequencing the amplified restriction fragments, and
• (f) comparing the sequence of the amplified restriction fragments with the sequence of the same restriction fragments from a reference source.

A method for detecting a change in the pattern or amount of RNA expression in a tissue or cell associated with an internal or external factor comprising:

• (1) determining the pattern of RNA expression in a first tissue or cell sample not subject to the internal or external change by a method comprising
• (a) reverse transcribing an RNA population to provide double-stranded cDNA;
• (b) digesting said double-stranded cDNA prepared from said first sample with one or more restriction endonucleases having a degenerate recognition or cleavage sequence, wherein the degenerate recognition or cleavage sequence is represented by the formula of Nm, where N is the extent of degeneracy, m is the number of degenerate bases, and m is 1-5, to produce restriction fragments having Nm different single-stranded overhangs for each restriction endonuclease;
• (c) ligating said restriction fragments to a series of adapters lacking restriction endonuclease sites, each adapter having a sequence complementary to one of said overhangs such that restriction fragments having identical overhangs are ligated to the same adapter;
• (d) amplifying said restriction fragments for no more than 25 cycles;
• and
• (e) determining the pattern of RNA expression in said first sample;
• (2) determining the pattern or amount of RNA expression in a second tissue or cell subject to the internal or external factor by performing the steps (1)(a)-(e) with said second tissue or cell;
• and
• (3) comparing said first and said second patterns or amounts to determine the effect of the internal or external factor on the pattern of RNA expression in the tissue or cell.

A method for diagnosis of a disease, condition or disorder comprising:

• (1) determining the pattern or amount of RNA expression in a first tissue or cell obtained from an organism free of said disease, condition or disorder by a method comprising
• (a) reverse transcribing an RNA population to provide double-stranded cDNA;
• (b) digesting said double-stranded cDNA prepared from said first sample with one or more restriction endonucleases having a degenerate recognition or cleavage sequence, wherein the degenerate recognition or cleavage sequence is represented by the formula of Nm, where N is the extent of degeneracy, and m is the number of degenerate bases, and wherein m is 1-5, to produce restriction fragments having Nm different single-stranded overhangs for each restriction endonuclease;
• (c) ligating said restriction fragments to a series of adapters lacking restriction endonuclease sites, each adapter having a sequence complementary to one of said overhangs such that restriction fragments having identical overhangs are ligated to the same adapter;
• (d) amplifying said restriction fragments for no more than 25 cycles;
• and
• (e) determining the pattern of RNA expression in said first sample;
• (2) determining the pattern or amount of RNA expression in a second tissue or cell obtained from an organism having said disease, condition or disorder by performing the steps (1)(a)-(e) with said second tissue or cell;
• (3) comparing said first and said second patterns or amounts to determine the effect of said disease, condition or disorder on the pattern or amount of RNA expression in the tissue or cell;
• and
• (4) if said disease, condition or disorder results in a change in the pattern or amount of RNA expression, using said change as an indication of the presence of said disease, condition or disorder, and the absence of said change as an indication of the absence of said disease, condition, or disorder. tissue or cell.