COMPOSITIONS AND METHODS FOR DIRECTIONAL NUCLEIC ACID AMPLIFICATION AND SEQUENCING

• US 20140303000A1
• Filed: 10/19/2012
• Published: 10/09/2014
• Est. Priority Date: 10/19/2011
• Status: Active Grant

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Armour, Chris
• Granted Patent: US 9,206,418 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12N 15/1017 by filtration, e.g. using filters, frits, membranes
• 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: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions

Abstract

The invention provides methods and compositions, including kits, for directional nucleic acid amplification and sequencing. The invention further provides methods and compositions for the construction of directional cDNA libraries.

Claims

1. A method for construction of a directional cDNA library comprising:

a. reverse transcribing a RNA sample to generate a first strand cDNA;

b. generating a second strand cDNA from the first strand cDNA, wherein at least one of the four dNTPs dATP, dCTP, dGTP or dTTP is replaced by a modified dNTP during second strand synthesis and incorporated into the second strand, thereby generating a double-stranded cDNA;

c. performing end repair on the double-stranded cDNA;

d. ligating adaptors to the double-stranded cDNA, wherein only one of the adaptors has the modified dNTP incorporated into a ligation strand of the adaptor;

e. performing gap repair; and

f. selectively cleaving the second strand and the adaptor that has the modified dNTP by a suitable cleavage agent, thereby generating a directional cDNA library comprising the first strand cDNA.

2. The method of claim 1, wherein the modified dNTP comprises dUTP.

3. The method of claim 1, wherein the method, optionally, further comprises fragmenting the double-stranded cDNA prior to step (c).

4. The method of claim 1, wherein step (f) comprises cleaving a base portion of the modified nucleotide, thereby forming an abasic site.

5. The method of claim 4, further comprising cleaving a phosphodiester backbone at the abasic site.

6. The method of claim 4, wherein the cleaving the base portion of the modified nucleotide comprises use of an enzyme.

7. The method of claim 5, wherein the cleaving the phosphodiester backbone at the abasic site comprises use of an enzyme, chemical agent, and/or heat.

8. The method of claim 7, wherein the enzyme is an endonuclease.

9. The method of claim 8, wherein the endonuelease is an apurinic/apyrimidinic endonuclease (APE).

10. The method of claim 6, wherein the enzyme comprises a glycosylase.

11. The method of claim 10, wherein the glycosylase is uracil-N-glycosylase (UNG) or uracil DNA glycosylase (UDG).

12. The method of claim 7, wherein the chemical agent comprises a primary amine.

13. The method of claim 7, wherein the chemical agent comprises a polyamine.

14. The method of claim 13, wherein the polyamine comprises N,N-dimethylethylenediamine (DMED).

15. The method of claim 1, wherein the cleavage agent comprises a glycosylase and a polyamine.

16. The method of claim 1, wherein the cleavage agent comprises a glycosylase and a APE.

17. The method of claim 1, further comprising amplifying the directional cDNA library, thereby generating amplified products.

18. The method of claim 17, wherein the amplification of the directional cDNA library comprises polymerase chain reaction (PCR), strand displacement amplification (SDA), multiple displacement amplification (MDA), rolling circle amplification (RCA), single primer isothermal amplification (SPIA), or ligase chain reaction (LCR).

19. The method of claim 18, wherein the amplification comprises PCR.

20. The method of claim 18, wherein the amplification comprises SPIA.

21. The method of claim 17, further comprising an additional step of sequencing the amplified products.

22. The method of claim 21, wherein the sequencing comprises next generation sequencing.

23. The method of claim 1, further comprising cleaving the RNA sample following reverse transcription.

24. The method of claim 23, wherein cleaving the RNA sample comprises exposing the RNA sample to an RNase following reverse transcription.

25. The method of claim 24, wherein the RNase is RNase H.

26. The method of claim 23, wherein cleaving the RNA sample comprises exposing the RNA sample to heat or chemical treatment or a combination thereof.

27. The method of claim 1 further comprising reducing or depleting non-desired nucleic acid sequences from the directional cDNA library.

28. The method of claim 27, wherein the non-desired nucleic acid is ribosomal RNA (rRNA).

29-62. -62. (canceled)

63. A method for construction of a directional cDNA library comprising:

a. synthesizing first and second strand cDNA from template RNA, wherein either first strand cDNA or second strand cDNA synthesis comprises incorporating a modified dNTP, to generate double stranded cDNA;

b. performing end repair on the double-stranded cDNA;

c. ligating adaptors to the double-stranded cDNA, wherein only one of the adaptors has the modified dNTP incorporated into a ligation strand of the adaptor;

d. performing gap repair; and

e. selectively cleaving the first or the second strand cDNA and the adaptor that has the modified dNTP by a suitable cleavage agent, thereby generating a directional cDNA library comprising the first or the second strand cDNA.