CDNA SYNTHESIS USING NON-RANDOM PRIMERS
The present invention provides methods for selectively amplifying a target population of nucleic acid molecules in a population of RNA template molecules (e.g., all mRNA molecules expressed in a cell type except for the most highly expressed mRNA species). The invention also provides a method of generating a population of oligonucleotide primers for transcriptome profiling of total RNA from a subject of interest.
1 . A method of generating a cDNA library representative of the transcriptome profile contained in total RNA in a subject of interest, comprising:
(a) synthesizing a population of single-stranded primer extension products from a target population of nucleic acid molecules within total RNA obtained from a subject of interest using reverse transcriptase enzyme and a first population of oligonucleotide primers comprising a hybridizing portion consisting of 6 to 9 nucleotides, a first PCR primer binding site located 5′ to the hybridizing portion, and a spacer portion consisting of from 2 to 10 nucleotides located between the hybridizing region and the PCR primer binding site, wherein the hybridizing portion is selected from all possible oligonucleotides having a length of from 6 to 9 nucleotides that hybridize under defined conditions to non-redundant target population of nucleic acid molecules, and do not hybridize under defined conditions to the non-target redundant population of nucleic acid molecules in the sample; and
(b) synthesizing double-stranded cDNA from the population of single-stranded primer extension products generated according to step (a) using a DNA polymerase and a second population of oligonucleotide primers comprising a hybridizing portion consisting of from 6 to 9 nucleotides, a second PCR primer binding site located 5′ to the hybridizing portion, and a spacer portion consisting of from 2 to 10 nucleotides located between the hybridizing portion and the PCR primer binding region, to generate a cDNA library representative of the transcriptome profile of the subject of interest.
2 . The method of claim 1 , further comprising PCR amplifying the double-stranded cDNA synthesized according to step (b) using a first PCR primer that binds to the first PCR primer binding site and a second PCR primer that binds to the second PCR primer.
3 . The method of claim 1 , further comprising cloning the double-stranded cDNA products into a vector to generate a cDNA library representative of the transcriptome profile of the subject of interest.
4 . The method of claim 1 , wherein the total RNA is obtained from a mammalian subject, and wherein the non-target population of nucleic acid molecules consists essentially of ribosomal RNA of the same species as the mammalian subject.
5 . The method of claim 1 , wherein the total RNA is obtained from a bacterial species, and wherein the non-target population of nucleic acid molecules consists essentially of ribosomal RNA of the same, or a related bacterial species.
6 . The method of claim 1 , wherein the sample contains blood obtained from a human subject infected with a parasite, and wherein the non-target population of nucleic acid molecules consists essentially of human globin RNA, human ribosomal RNA and ribosomal RNA from the same species of parasite that is present in the sample.
7 . The method of claim 1 , further comprising sequencing at least a portion of the cDNA library.
8 . The method of claim 1 , wherein the population of hybridizing portions in the first population of oligonucleotide primers is selected from all possible oligonucleotides having a length of 6 nucleotides that do not hybridize under defined conditions to the non-target redundant nucleic acid molecules in the population of RNA template molecules.
9 . The method of claim 1 , wherein the spacer region contained in at least one of the first population of oligonucleotide primers or the second population of oligonucleotide primers consists of 6 random nucleotides.
10 . The method of claim 1 , wherein the spacer region contained in the first population of oligonucleotide primers and the second population of oligonucleotide primers consists of 6 random nucleotides.
11 . A kit for selectively amplifying a target population of nucleic acid molecules, the kit comprising:
(i) a first reagent comprising a first population of oligonucleotides for first strand cDNA synthesis, wherein each oligonucleotide in the first population of oligonucleotides comprises a hybridizing portion, a defined sequence portion located 5′ to the hybridizing portion, and a spacer region consisting of 6 random nucleotides located between the hybridizing portion and the defined sequence portion, wherein the hybridizing region is a member of the population of oligonucleotides comprising SEQ ID NOS:1-749; and
(ii) a second reagent comprising a second population of oligonucleotides for second strand cDNA synthesis, wherein each oligonucleotide in the second population of oligonucleotides comprises a hybridizing portion, a defined sequence portion located 5′ to the hybridizing portion, and a spacer region consisting of 6 random nucleotides located between the hybridizing portion and the defined sequence portion, wherein the hybridizing portion is a member of the population of oligonucleotides comprising SEQ ID NOS:750-1498.
12 . The kit of claim 11 , wherein the population of hybridizing portions in the first population of oligonucleotides comprises the oligonucleotides consisting of SEQ ID NOS:1-749, and wherein the population of hybridizing portions in the second population of oligonucleotides comprises the oligonucleotides consisting of SEQ ID NOS:750-1498.
13 . The kit of claim 11 , further comprising at least one of the following components: a reverse transcriptase, a DNA polymerase, a DNA ligase, a RNase H enzyme, a Tris buffer, a potassium salt, a magnesium salt, an ammonium salt, a reducing agent, deoxynucleoside triphosphates, or a ribonuclease inhibitor.
14 . A method of generating a population of oligonucleotide primers for transcriptome profiling of total RNA from a subject of interest, the method comprising:
(a) providing a first population of oligonucleotide primers, each primer comprising a hybridizing portion consisting of 6 to 9 nucleotides, and a first primer binding site located 5′ to the hybridizing portion;
(b) synthesizing a population of single-stranded primer extension products from the total RNA of a subject of interest using reverse transcriptase enzyme and the first population of oligonucleotide primers of step (a);
(c) synthesizing double-stranded cDNA from the population of single-stranded primer extension products generated according to step (b);
(d) sequencing a portion of the double-stranded cDNA products generated according to step (c) and identifying the subset of primers containing hybridizing regions that primed cDNA synthesis from unwanted redundant RNA sequences that are present at a frequency greater than a minimum threshold level of from 0.5% to 2% of the total sequences analyzed; and
(e) modifying the first population of oligonucleotide primers to exclude the subset of primers identified in step (d) to generate a second population of oligonucleotide primers for transcriptome profiling of the total RNA from the sample of interest.
15 . The method of claim 14 , wherein the population of hybridizing portions of the first population of oligonucleotide primers is selected from all possible oligonucleotides having a length of 6 nucleotides.
16 . The method of claim 15 , wherein the population of hybridizing portions is further selected by comparing the reverse complement of each 6 nucleotide hybridizing region to the nucleotide sequences of ribosomal RNA from same species as the subject of interest and eliminating all primers comprising hybridizing portions that have a perfect match to the ribosomal RNA sequences from the population of oligonucleotide primers prior to use in step (b).
17 . The method of claim 14 , wherein the subject of interest is a mammalian subject.
18 . The method of claim 14 , wherein the subject of interest is a bacterial species.
19 . The method of claim 14 , further comprising carrying out steps (b) and (c) with the second population of oligonucleotide primers generated according to step (e), to generate a third population of oligonucleotide primers.
20 . The method of claim 14 , further comprising synthesizing a population of single-stranded primer extension products from total RNA from the subject of interest using reverse transcriptase enzyme and the second population of oligonucleotide primers of step (e).
21 . The method of claim 14 , wherein the first population of oligonucleotide primers further comprises a spacer portion consisting of from 2 to 10 random nucleotides located between the hybridizing region and the primer binding site.
22 . The method of claim 14 , wherein the first population of oligonucleotide primers further comprises a spacer portion consisting of 6 random nucleotides located between the hybridizing region and the primer binding site.