Methods for producing stranded cDNA libraries

• US 9,255,265 B2
• Filed: 03/15/2013
• Issued: 02/09/2016
• 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): Leavitt, Maria
• Assistant Examiner(s): Bunker, Amy M
• Application Number:
  US13/835,757
• Publication Number:
  US 20140274807A1
• Time in Patent Office: 1,061 Days
• Field of Search: 506/9, 506/23
• US Class Current: 1/1
• 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 creating a double-stranded cDNA library representing a particular single stranded RNA molecule in an RNA sample comprising:

(a) hybridizing a plurality of first primers to each of said single stranded RNA molecules in an RNA sample under conditions wherein complexes are formed between the 3′

region of each of two or more first primers in said plurality of first primers and each of two or more RNA molecules in said RNA sample, wherein said 3′

region of said first primers comprise a random nucleotide sequence and a first nucleotide sequence tag;

(b) extending said plurality of first primers complexed to each of said RNA in an RNA sample by reverse transcription in the 3′

to 5′

direction relative to the single stranded RNA molecule and subsequently removing the two or more RNA molecules, thereby generating two or more single stranded complementary DNA (cDNA) molecules of said two or more RNA molecules, wherein said 3′

region of said generated two or more single stranded cDNA molecules comprises said random nucleotide sequence and said first nucleotide sequence tag;

(c) hybridizing a plurality of double stranded polynucleotide molecules comprising a second nucleotide sequence tag to said two or more generated single stranded cDNA molecules, wherein a double stranded polynucleotide molecule of said plurality of double stranded polynucleotide molecules comprises a first strand and a complementary second strand, said first strand comprises a second nucleotide sequence tag and a 3′

overhang comprising a second random nucleotide sequence, and wherein said second strand comprises a 5′

phosphate group, under conditions wherein;

(i) a complex is formed between said 3′

overhang of a double stranded polynucleotide molecule in said plurality of double stranded polynucleotide molecules and a 3′

region of said two or more generated single stranded cDNA molecules, and

(ii) a 5′

end of said complementary second strand of said double stranded polynucleotide molecule in said plurality of double stranded polynucleotide molecules is adjacent to a 3′

end of said two or more generated single stranded cDNA molecules;

(d) attaching said 5′

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

end of said two or more generated cDNA molecules forming a covalent bond, thereby leaving said first strand of said double stranded polynucleotide molecule unattached to said generated two or more cDNA molecules;

(e) removing said unattached strands of said double stranded polynucleotide molecules, thereby forming two or more single stranded cDNA molecules comprising a first and a second nucleotide sequence tag; and

(f) converting said two or more single stranded cDNA molecules to double stranded cDNA molecules, thereby creating a double-stranded cDNA library representing a particular single stranded RNA molecule in an RNA sample.

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

3. The method of claim 1, wherein said second random nucleotide sequence of said plurality of double stranded polynucleotide molecules consists of 6 to 9 nucleotides.

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

overhang of said double stranded polynucleotide molecules comprise a 3′

block.

5. The method of claim 4, wherein said 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 said removing step comprises cleaving said two or more RNA molecules with RNase H.

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

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

end of said cDNA molecules.