Methods for producing stranded cDNA libraries

• US 10,047,359 B2
• Filed: 01/12/2016
• Issued: 08/14/2018
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Stephens, Kathryn M., Burch, Marcus
• Primary Examiner(s): Kim, Young J
• Application Number:
  US14/993,908
• Publication Number:
  US 20160194628A1
• Time in Patent Office: 945 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

The present system provides novel methods and compositions for selecting a particular strand of RNA and/or producing a cDNA library that results in an unbiased representation of RNA in a sample.

Claims

1. A method for selecting a particular single-stranded RNA molecule in an RNA sample comprising:

(a) hybridizing a first primer to the single-stranded RNA molecule in an RNA sample under conditions wherein a complex is formed between the 3′

region of the first primer and the single-stranded RNA molecule in the RNA sample, wherein the 3′

region of the first primer comprises a first a first random nucleotide sequence and a first nucleotide sequence tag;

(b) extending the first primer complexed to the 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 the single-stranded RNA molecule;

(c) hybridizing a double stranded polynucleotide molecule comprising a second nucleotide sequence tag to the cDNA molecule, wherein the double stranded polynucleotide molecule comprises a first strand and a complementary second strand, and wherein the 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 the double stranded polynucleotide molecule and the 3′

region of the cDNA molecule, and

(ii) the 5′

end of the complementary second strand of the double stranded polynucleotide molecule is adjacent to the 3′

end of the cDNA molecule;

(d) attaching the 5′

end of the complementary second strand of the double stranded polynucleotide molecule to the 3′

end of said cDNA molecule forming a covalent bond, thereby leaving the first strand of the double stranded polynucleotide molecule unattached to the cDNA molecule;

(e) removing the unattached strand of the 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 the RNA molecule by hybridizing a probe or a second primer to the first or second nucleotide sequence tags.

2. The method of claim 1, wherein the random nucleotide sequence of said first primer consists of 6 to 9 nucleotides.

3. The method of claim 1, wherein the random nucleotide sequence of the double stranded polynucleotide molecule consists of 6 to 9 nucleotides.

4. The method of claim 1, wherein the 3′

overhang of the double stranded polynucleotide molecule comprises a 3′

block.

5. The method of claim 4, wherein the 3′

block is a phosphorylated 3′

end of said 3′

overhang, an RNA nucleotide, a C3 spacer, or an amine group (NH₂).

6. The method of claim 1, wherein step (b) further comprises removing the RNA molecule.

7. The method of claim 6, wherein the removing comprises cleaving the RNA molecule with RNase H.

8. The method of claim 1, wherein step (d) comprises ligating the 5′

end of the complementary second strand of the double stranded polynucleotide molecule to the 3′

end of the cDNA molecule.

9. The method of claim 1, wherein step (f) comprises hybridizing the second primer to the second nucleotide sequence tag.

10. The method of claim 9, wherein selecting comprises extending the second primer.

11. The method of claim 10, wherein the extension of the second primer comprises incorporation of a detectable label.

12. The method of claim 1, wherein the probe or second primer of step (f) is immobilized to a solid support.