METHOD OF PRODUCING DNA AND DNA LIBRARY

• US 20190323005A1
• Filed: 04/19/2018
• Published: 10/24/2019
• Est. Priority Date: 04/19/2018
• Status: Active Grant

• Current Assignee: City University of Hong Kong
• Original Assignee: City University of Hong Kong
• Inventors: Kwok, Chun Kit, Yeung, Pui Yan
• Granted Patent: US 11,085,042 B2
• Time in Patent Office: Days
• CPC Class Codes: C02F 1/469 by electrochemical separation, e.g. by electro-osmosis, electrodialysis, electrophoresis
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

A method of producing a cDNA from a sample of nucleic acids includes the steps of: generating a dephosphorylated RNA from the sample; ligating the dephosphorylated RNA with a first adapter in the presence of a crowding agent to produce a ligated product; removing the excess first adapter by adding at least two enzymes; and performing reverse transcription in a lithium-containing buffer to produce the cDNA. A method of preparing a DNA library and a kit for such preparation are also disclosed.

Claims

1. A method of producing a cDNA from a sample of nucleic acids, comprising the steps of:

a) generating a dephosphorylated RNA from the sample;

b) ligating the dephosphorylated RNA with a first adapter in the presence of a crowding agent to produce a ligated product;

c) removing the excess first adapter by adding at least two enzymes; and

d) performing reverse transcription in a lithium-containing buffer to produce the cDNA.

2. The method of claim 1, wherein the step a) comprises:

adding a dephosphorylating agent comprising one or more alkaline phosphatases to the sample; and

incubating the mixture under which dephosphorylation takes place.

3. The method of claim 2, wherein the dephosphorylating agent comprises shrimp alkaline phosphatase, FastAP Thermosensitive Alkaline Phosphatase, or a combination thereof.

4. The method of claim 1, wherein the crowding agent comprises polyethylene glycol.

5. The method of claim 1, wherein in the step b), the ratio of the amount of the dephosphorylated RNA to the amount of the first adapter is about or less than 1:

2.5.

6. The method of claim 5, wherein the ratio of the amount of the dephosphorylated RNA to that of the first adapter is about or less than 1:

5.

7. The method of claim 1, wherein the step c) comprises:

adding a deadenylase and a 5′

-3′

DNA exonuclease to the ligated product, and incubating the mixture for at least 15 minutes at about 20-35°

C.

8. The method of claim 7, wherein the step c) further comprises purification after the incubation, wherein the purification is performed by using a purification column.

9. The method of claim 1, wherein the lithium-containing buffer comprises lithium chloride, lithium acetate, lithium borate, or a combination thereof.

10. The method of claim 1, wherein in the step d), a locked nucleic acid is used in the reverse transcription to produce the cDNA.

11. A method of preparing a DNA library, comprising the steps of:

i) producing a cDNA from a sample according to the method of claim 1; and

ii) ligating the cDNA with a second adapter in the presence of a crowding agent to produce a ligated product; and

amplifying the ligated product.

12. The method of claim 11, wherein the ratio of the amount of the cDNA to that of the second adapter is less than 1:

2.5.

13. The method of claim 12, wherein the ratio of the amount the cDNA to that of the second adapter is about or less than 1:

5.

14. The method of claim 11, wherein the crowding agent has a concentration of about 1% to 20% (w/w).

15. The method of claim 11, wherein the crowding agent comprises PEG 8,000, PEG 6,000 or a combination thereof.

16. The method of claim 11, wherein the second adapter comprises a sequence represented by SEQ ID NO:

2.

17. The method of claim 11, wherein the ligation is conducted at a temperature of about 20-40°

C. for at least 1 h.

18. A kit for preparing a DNA library from a sample of nucleic acids, comprising:

a dephosphorylating agent comprising one or more alkaline phosphatases;

a crowding agent; and

a mixture comprising a deadenylase, a 5′

-3′

exonuclease, or a combination thereof.

19. The kit of claim 18, further comprises a first adapter comprising a SEQ ID NO:

1.

20. The kit of claim 18, further comprises a second adapter comprising a sequence represented by SEQ ID NO:

2.

21. The kit of claim 18, wherein the dephosphorylating agent comprises shrimp alkaline phosphatase, FastAP alkaline phosphatase, or a combination thereof.

22. The kit of claim 18, further comprises a locked nucleic acid.