Multiplex end-tagging amplification of nucleic acids

Patent metadata

Field	Value
Publication number	US-11530436-B2
Application number	US-201816615872-A
Country	US
Kind code	B2
Filing date	May 23, 2018
Priority date	May 23, 2017
Publication date	Dec 20, 2022
Grant date	Dec 20, 2022

Abstract

The present disclosure provides a method for assembly of genomic DNA using multiplex end-tagging amplification of genomic fragments.

First claim

What is claimed is:

1. A method of DNA amplification comprising
contacting genomic DNA with a library of transposomes with each transposome of the library having two transposases and two transposon DNA, wherein each transposon DNA includes a transposase binding site and a primer binding site sequence, wherein the primer binding site sequence is different from the primer binding site of other members of the transposome library, and wherein each transposome within the library of transposomes includes two different primer binding site sequences,
wherein the library of transposomes bind to target locations along the genomic DNA and the transposase cleaves the genomic DNA into a plurality of double stranded genomic DNA fragments representing a genomic DNA fragment library, with each double stranded genomic DNA fragment includes a unique and/or different primer binding site sequence on each end of the genomic DNA fragment,
filling a gap between the transposon DNA and the genomic DNA fragment to form a library of double stranded genomic DNA fragment extension products having unique and/or different primer binding site sequences at each end, and
amplifying the double stranded genomic DNA fragment extension products to produce amplicons.

2. The method of claim 1 further including sequencing the amplicons.

3. The method of claim 1 wherein the genomic DNA is whole genomic DNA obtained from a single cell.

4. The method of claim 1 wherein the transposase is Tn5 transposase, Mu transposase, Tn7 transposase or IS5 transposase.

5. The method of claim 1 wherein the transposon DNA includes a double-stranded 19 bp Tnp binding site and an overhang, wherein the overhang includes a unique and/or different primer binding site sequence at the 5′ end of the overhang.

6. The method of claim 1 wherein bound transposases are removed from the double stranded fragments before gap filling and extending of the double stranded genomic DNA fragments.

7. The method of claim 1 wherein the genomic DNA is from a prenatal cell.

8. The method of claim 1 wherein the genomic DNA is from a cancer cell.

9. The method of claim 1 wherein the genomic DNA is from a circulating tumor cell.

10. The method of claim 1 wherein the genomic DNA is from a single prenatal cell.

11. The method of claim 1 wherein the genomic DNA is from a single cancer cell.

12. The method of claim 1 wherein the genomic DNA is from a single circulating tumor cell.

13. The method of claim 1 where the genomic DNA is the product of chromatin conformation capture from a single cell or a small sample.

14. The method of claim 1 where the genomic DNA is the native or fixed chromatin from a single cell or minute amount of samples.

15. The method of claim 1 wherein the unique and different primer binding site sequence is a specific PCR primer binding site.

16. The method of claim 1 wherein the library of transposomes includes 1 to 100 unique and different primer binding site sequences.

17. The method of claim 1 wherein the library of transposomes includes 1 to 10 unique and different primer binding site sequences.

18. The method of claim 1 wherein the library of transposomes includes 5 to 50 unique and different primer binding site sequences.

19. The method of claim 1 wherein the library of transposomes includes 30 to 100 unique and different primer binding site sequences.

20. The method of claim 1 wherein the library of transposomes includes 15 to 25 unique and different primer binding site sequences.

21. The method of claim 1 wherein the library of transposomes includes 100 to 1,000 unique and different primer binding site sequences.

22. The method of claim 1 wherein the library of transposomes includes 1,000 to 10,000 unique and different primer binding site sequences.

23. The method of claim 1 wherein the library of transposomes includes 10,000 to 100,000 unique and different primer binding site sequences.

24. The method of claim 1 wherein the different primer binding site sequences are orthogonal.

Assignees

• Harvard College

Inventors

• Xie Xiaoliang Sunney
• Xing Dong
• Chang Chi-Han
• Tan Longzhi

Classifications

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/6806 Primary

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

• C12Q2521/507

  Recombinase · CPC title

• C12N15/1082

  Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors · CPC title

• C12Q2525/155

  incorporating/generating a new priming site · CPC title