| 主权项 |
1. A method for selecting a particular single-stranded RNA molecule in an RNA sample comprising:
(a) hybridizing a first primer to said single-stranded RNA molecule in an RNA sample under conditions wherein a complex is formed between the 3′ region of said first primer and said single-stranded RNA molecule in said RNA sample, wherein said 3′ region of said first primer comprises a first random nucleotide sequence and a first nucleotide sequence tag; (b) extending said first primer complexed to said single-stranded RNA molecule by reverse transcription in the 3′ to 5′ direction relative to the single-stranded RNA molecule, thereby generating a complementary DNA (cDNA) molecule of said single-stranded RNA molecule; (c) hybridizing a double stranded polynucleotide molecule comprising a second nucleotide sequence tag to said cDNA molecule, wherein the double stranded polynucleotide molecule comprises a first strand and a complementary second strand, and wherein said first strand comprises a 3′ overhang comprising a second random nucleotide sequence, under conditions wherein:
(i) a complex is formed between the 3′ overhang of said double stranded polynucleotide molecule and the 3′ region of said cDNA molecule, and(ii) the 5′ end of the complementary second strand of said double stranded polynucleotide molecule is adjacent to the 3′ end of said cDNA molecule; (d) attaching said 5′ end of said complementary second strand of said double stranded polynucleotide molecule to said 3′ end of said cDNA molecule forming a covalent bond, thereby leaving said first strand of said double stranded polynucleotide molecule unattached to said cDNA molecule; (e) removing said unattached strand of said double stranded polynucleotide molecule, thereby forming a single stranded cDNA molecule comprising a first and a second nucleotide sequence tag; and (f) selecting for a particular cDNA strand of said RNA molecule by hybridizing a probe or a second primer to said first or second nucleotide sequence tags. |
