Patent ID: 6623965
Filing Date: 2003-09-23
Classification: C12N

Abstract:
A method of cloning DNA comprising:(a) priming poly A+ mRNA to generate (âˆ’) first strand cDNAs; (b) homopolymerically tailing the (âˆ’) first strand to form a heteroduplex; (c) denaturing the heteroduplex; (d) removing the mRNA to yield a single-stranded (âˆ’) cDNA; (e) mixing the single-stranded (âˆ’) cDNA with a first oligonucleotide, said oligonucleotide comprising a restriction site, to produce annealed, short, double-stranded DNA segments corresponding to positions of the restrictions site on the single-stranded (âˆ’) cDNA; (f) cleaving the double-stranded DNA with a restriction enzyme specific to the restriction sites to yield single-strand cDNAs bound on their 5â€² end by a sticky end left by the restriction enzyme and on their 3â€² end by a poly-dG or poly-dC tract; (g) annealing a second oligonucleotide comprising nucleotides complementary to the 3â€² end of the single-stranded cDNAs and containing the same restriction site as in the first oligonucleotide to the 3â€² end of the single-stranded cDNA so that the 5â€² end of the single-stranded cDNAs can loop back and also anneal to the second oligonucleotide at the restriction site to form primed and gapped single-stranded (âˆ’) cDNAs; and (h) adding DNA polymerase and DNA ligase to the primed and gapped single-stranded (âˆ’) cDNAs.