Multiplex End-Tagging Amplification of Nucleic Acids

Patent metadata

Field	Value
Publication number	US-2023203563-A1
Application number	US-202218055024-A
Country	US
Kind code	A1
Filing date	Nov 14, 2022
Priority date	May 23, 2017
Publication date	Jun 29, 2023
Grant date	—

Abstract

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

First claim

1 .- 26 . (canceled)

27 . A method of creating double stranded DNA amplicons having unique and/or different priming site sequences at each end comprising
separating a target double stranded DNA having transposase binding sequence and the same priming site sequence at each end into a first single strand and second strand, annealing to the first strand, a first primer having a first sequence complementary to the transposase binding site and a second sequence noncomplementary to the priming site sequence, annealing to the second strand, a second primer having a first sequence complementary to the transposase binding site and a second sequence complementary to the priming site sequence, extending the first primer along the first strand and extending the second primer along the second strand and amplifying the extension products to produce double stranded DNA amplicons having unique and/or different priming site sequences at each end.

28 . A method of amplifying two strands of a double stranded nucleic acid sequence having different priming sites at each end comprising
separating the double stranded nucleic acid sequence into a first strand and a second strand, amplifying the first strand in the absence of the second strand to create first strand amplicons, amplifying the second strand in the absence of the first strand to create second strand amplicons, sequencing the first strand amplicons, and sequencing the second strand amplicons.

29 . The method of claim 28 further comprising
annealing the 3′-end of the first strand and second strand with primers containing a priming region which is complementary to the 3′-end of the first strand and the second strand and a first adapter sequence, synthesizing complementary strands by a DNA polymerase, removing the excess primers with an exonuclease,
annealing the 3′-end of the synthesized, complementary strands of the first strand and the second strand with primers containing a priming region which is complementary to the 3′-end of the first strand and the second strand and a second adapter sequence, synthesizing complementary strands by a DNA polymerase, removing the excess primers with an exonuclease,
amplifying the target sequences by PCR with primers which anneal to the first adapter sequence and second adapter sequence to create amplicons for the first strand and the second strand,
sequencing the amplicons to distinguish the first strand from the second strand, wherein the first end of the first strand is tagged with the first adapter, the second end of the first strand is tagged with the second adapter,
wherein the first end of the second strand is tagged with the second adapter, the second end of the second strand is tagged with the first adapter.

30 . The method of claim 15 where chromatin conformation capture is from a diploid sample, and haplotype information of each captured chromatin contact is determined.

Assignees

• Harvard College

Inventors

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

Classifications

• C12Q2521/101

  DNA polymerase · CPC title

• C12Q2525/155

  incorporating/generating a new priming site · CPC title

• C12Q1/6806 Primary

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

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title