Directional amplification of RNA

Patent metadata

Field	Value
Publication number	US-10711296-B2
Application number	US-201615550559-A
Country	US
Kind code	B2
Filing date	Mar 24, 2016
Priority date	Mar 24, 2015
Publication date	Jul 14, 2020
Grant date	Jul 14, 2020

Abstract

Processes, oligonucleotides, and kits for amplifying RNA. In particular, the processes generate and amplify cDNA libraries in which the orientation of the input RNA molecule is preserved in the products. Among the various aspects of the present disclosure is the provision of process for directionally amplifying RNA. The process comprises reverse transcribing at least one RNA molecule in the presence of a plurality of first synthesis primers to generate a plurality of first strands of complementary DNA (cDNA), wherein each of the first synthesis primers comprises a 3′ sequence having complementarity to a portion of the RNA molecule, a non-complementary 5′ sequence corresponding to one or more amplification primers, and optionally an internal tag sequence comprising a first tag sequence.

First claim

What is claimed is:

1. A process for directionally amplifying RNA, the process comprising:
(a) reverse transcribing at least one RNA molecule in the presence of a plurality of first synthesis primers to generate a plurality of first strands of complementary DNA (cDNA), each of the first synthesis primers comprising a 3′ sequence having complementarity to a portion of the RNA molecule, a 5′ sequence corresponding to one or more methylated amplification primers, and optionally an internal tag sequence comprising a first tag sequence;
(b) replicating the plurality of first strands of cDNA in the presence of a plurality of second synthesis primers to generate a plurality of double-stranded cDNA products, each of the second synthesis primers comprising a 3′ sequence having complementarity to a portion of the first strands of cDNA, a 5′ sequence corresponding to the one or more methylated amplification primers, and optionally an internal tag sequence comprising a second tag sequence, provided that either one or both of the pluralities of first and second synthesis primers comprises the internal tag sequence such that each double-stranded cDNA product is flanked by either the first or second tag sequence or both the first and second tag sequences; and
(c) amplifying the plurality of double-stranded cDNA products in the presence of the one or more methylated amplification primers to generate an amplified library of cDNA products;
wherein the 5′ sequence of the first and second synthesis primers further comprises at least one binding element for a methylation-dependent restriction enzyme, and
wherein the amplified library of cDNA products is contacted with the methylation-dependent restriction enzyme.

2. The process of claim 1 , wherein the 3′ sequence of the first and second synthesis primers is random, semi-random, or a combination thereof.

3. The process of claim 1 , wherein the binding element is hemi-methylated.

4. The process of claim 1 , wherein the 5′ sequence of the first and second synthesis primers comprises a majority of non-complementary nucleotides.

5. The process of claim 1 , wherein the first and/or second tag sequences are at least 6 nucleotides in length.

6. The process of claim 1 , wherein step (a) is conducted in the presence of a reverse transcriptase, deoxyribonucleotides, and optionally Actinomycin D.

7. The process of claim 1 , wherein step (a) is followed by contact with a 3′-5′ deoxyribonuclease to degrade unhybridized first synthesis primers.

8. The process of claim 1 , wherein step (b) commences with heat denaturation of the plurality of first strands of cDNA in the presence of the plurality of second synthesis primers, and step (b) is conducted in the presence of a non-strand-displacing DNA polymerase and deoxyribonucleotides.

9. The process of claim 1 , wherein step (b) optionally is followed by contact with a 3′-5′ deoxyribonuclease to degrade unhybridized second oligonucleotides.

10. The process of claim 1 , wherein step (c) commences with heat denaturation of the plurality of double-stranded cDNA products in the presence of the one or more amplification primers, and step (c) is conducted in the presence of a thermostable DNA polymerase and deoxyribonucleotides.

11. The process of claim 1 , further comprising incorporating adaptor sequences to the 5′ ends of the amplified library of cDNA products.

12. The process of claim 1 , which is devoid of one or more purification steps between steps (a) and (b) and/or between steps (b) and (c).

Assignees

• Sigma Aldrich Co Llc

Inventors

• Heuermann Kenneth B
• Kreader Carol A
• Robert Jaime K
• Ward Brian W

Classifications

• C12N15/1096

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Q2525/101

  incorporating non-naturally occurring nucleotides, e.g. inosine · CPC title