Compositions and methods for constructing strand specific cDNA libraries
Provided herein are compositions, kits and methods for the production of strand-specific cDNA libraries. The compositions, kits and methods utilize properties of double stranded polynucleotides, such as RNA-cDNA duplexes to capture and incorporate a novel sequencing adapter. The methods are useful transcriptome profiling by massive parallel sequence, such as full-length RNA sequencing (RNA-Seq) and 3′ tag digital gene expression (DGE).
1 . A method of generating a strand-specific DNA molecule from double stranded nucleic acid, the method comprising:
providing a double-stranded nucleic acid duplex comprising a first strand and a second strand;
without removal of the first strand or the second strand, annealing a partially double stranded oligonucleotide 5′ adapter to the 3′ end of the first strand in the duplex, wherein the 5′ adapter comprises:
(i) a first strand capturing oligonucleotide comprising at least 20 nucleotides and a 3′ overhang comprising about 6-12 consecutive random nucleotides that anneal to the 3′ end of the first strand; and
(ii) a second strand blocking oligonucleotide comprising at least 20 nucleotides complementary to at least a portion of the first strand capturing oligonucleotide; and
generating the strand specific DNA molecule by extending the first strand capturing oligonucleotide of the 5′ adapter using a DNA polymerase or a fragment thereof to generate a DNA strand complementary to the first strand.
2 . The method of claim 1 , wherein the double-stranded nucleic acid duplex is a double-stranded DNA.
3 . The method of claim 1 , wherein the providing comprises isolating the double-stranded nucleic acid duplex from a biological sample.
4 . The method of claim 3 , wherein the biological sample is an animal tissue sample.
5 . The method of claim 3 , wherein the biological sample is a plant tissue sample.
6 . The method of claim 1 , wherein the providing comprises isolating a single-stranded DNA or RNA from a biological sample and converting the single-stranded DNA or RNA into the double-stranded nucleic acid duplex with a polymerase.
7 . The method of claim 1 , further comprising amplifying the second strand using a primer complementary to the second strand blocking oligonucleotide.
8 . The method of claim 7 , wherein amplifying comprises polymerase chain reaction.
9 . The method of claim 1 , further comprising determining the sequence of the amplified second strand.
10 . The method of claim 1 , wherein the DNA polymerase or fragment thereof is DNA polymerase I.
11 . The method of claim 1 , wherein the DNA polymerase or fragment thereof is Klenow fragment.
12 . The method of claim 1 , wherein the second strand blocking oligonucleotide of the 5′ adapter is 5′ phosphorylated.
13 . The method of claim 12 , wherein the DNA polymerase is a Klenow fragment and a ligase.