Patent ID: 6300058
Filing Date: 2001-10-09
Classification: C07H,C12N,C12P,C12Q

Abstract:
A quantitative method for identifying probes and thereafter detecting and quantifying mRNA in a sample using said probes, without the need to purify mRNA from cells, said method comprising:(a) identifying an oligonucleotide sequence of no more than 40 nucleotides but no less than 17 nucleotides which is complementary to a sequence unique to said mRNA, which has a melting temperature (T.sub.m) within a preselected range with said mRNA at a selected sodium and formamide concentration, said T.sub.m being determined by the formula;T.sub.m =81.5-16.6(log[Na])-0.63%(formamide)+0.41(%(G+C))-600/N, wherein log[Na] is the log function of the sodium concentration, 0.63%(formamide) is the concentration of formamide, %(G+C) is the percentage of matched GC base pairs, and N is the probe length,said oligonucleotide sequence being identified by a method comprising:(i) retrieving a plurality of user-selected gene sequences including the gene sequence of said mRNA from a database and inputting said sequences into a computer system for calculating T.sub.m ;(ii) calculating the T.sub.m s of a plurality of candidate oligonucleotide sequences extracted from the gene sequence of said mRNA, at the selected sodium and formamide concentration with said mRNA, using said computer system;(iii) calculating the T.sub.m s of said plurality of candidate oligonucleotide sequences at the selected sodium and formamide concentration with each of said user-selected gene sequences other than that of said mRNA; and(iv) identifying said oligonucleotide sequence as the candidate sequence that has a T.sub.m within the preselected range with said mRNA and has the lowest T.sub.m overall with the other user-selected gene sequences;(b) immobilizing a specific probe having said oligonucleotide sequence to an insoluble support;(c) incubating said sample with said insoluble support under conditions wherein said nucleotide sequence unique to said mRNA will specifically hybridize with said specific probe, thereby immobilizing mRNA having said unique nucleotide sequence present in said sample to said insoluble support;(d) washing non-immobilized components of said sample from said insoluble support;(e) labeling mRNA on said support in a manner that the amount of label incorporated onto said support is related to the amount of mRNA on said support; and(f) measuring the amount of the label immobilized on said support.