Patent ID: 8497067

Claim:
A method of restoring nucleic acid sequences recovered fragmented or degraded from a tissue comprising: a) reverse transcribing mRNA from the tissue using random primers or a 5′-promoter-oligo-dT(10-30)-VN-3′ primer, or a combination of both, to obtain a RNA/DNA duplex made of mRNA and single-stranded cDNA that is complementary to the mRNA, wherein V is nucleotide A, C, or G; N is nucleotide A, C, G or T; and oligo-dT(10-30) is 10 to 30 deoxyribothymidines; b) removing RNA from the RNA/DNA duplex of step a) to yield a single-stranded cDNA primer that is complementary to the mRNA; c) purifying the single-stranded cDNA primer obtained in step b) to obtain a purified primer; d) hybridizing the purified single-stranded cDNA primer from step c) to a single stranded oligonucleotide to obtain a partially double-stranded oligonucleotide complex with a single-stranded cDNA portion specific to a gene expressed in the tissue, wherein if the primer in step a) is a 5′-promoter-oligo-dT(10-30)-VN-3′ primer, then the single stranded oligonucleotide in step d) is 5′-NB-oligo-dA(10-30)-complementary promoter-3′, wherein B is nucleotide C, G or T; oligo-dA(10-30) is 10 to 30 deoxyriboadenosines; the number of oligo-dAs is the same as the number of oligo-dTs in the reverse-transcribing primer in step a); and the complementary promoter is complementary to the promoter in step a); and wherein if the primers in step a) are random primers, then the single stranded oligonucleotides in step d) comprise a complementary promoter that is complementary to the promoter in step a), a dideoxynucleotide at their 3′ end, and the same numbers of nucleotides as the random primers in step a); e) annealing the single-stranded cDNA portion of the partially double-stranded oligonucleotide complex formed in step d) with sense RNA to form a hybrid product comprising single-stranded sense RNA and cDNA primer that is complementary-bound to the single-stranded sense RNA; f) reverse-transcribing the single-stranded sense RNA of the hybrid product obtained in step e) to extend and restore nucleic acid sequences on the cDNA primer that is complementary-bound to the single-stranded sense RNA to obtain a cDNA/sense RNA duplex; and g) synthesizing double-stranded DNA from the cDNA/sense RNA duplex obtained in step f) so as to obtain a double-stranded DNA that contains a restored nucleic acid sequence from the tissue.