Student or educator? Sign up with your school email and get 6 months of Pro free.
CoChat
Sign Up
Cited by 13

The DOI matches its Crossref record; 26 of the 26 references it cites were found in a registry or an index.

3′ Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3′OH ends in DNA or RNA

Wang, Lin, Xi, Yang, Zhang, Wenwei, Wang, Weimao, Shen, Hanjie, Wang, Xiaojue, Zhao, Xia, Alexeev, Andrei, Peters, Brock A, Albert, Alayna, Xu, Xu, Ren, Han, Wang, Ou, Kirkconnell, Killeen, Perazich, Helena, Clark, Sonya, Hurowitz, Evan, Chen, Ao, Xu, Xun, Drmanac, Radoje, Jiang, Yuan·2019·DNA Research
DOI 10.1093/dnares/dsy037PMC PMC6379041

Published version of a preprint

Open this paper in CoChat — free account

Abstract

Abstract Nucleic acid ligases are crucial enzymes that repair breaks in DNA or RNA during synthesis, repair and recombination. Various molecular tools have been developed using the diverse activities of DNA/RNA ligases. Herein, we demonstrate a non-conventional ability of T4 DNA ligase to join 5’ phosphorylated blunt-end double-stranded DNA to DNA breaks at 3’ recessive ends, gaps, or nicks to form a 3’ branch structure. Therefore, this base pairing-independent ligation is termed 3’ branch ligation (3’BL). In an extensive study of optimal ligation conditions, similar to blunt-end ligation, the presence of 10% PEG-8000 in the ligation buffer significantly increased ligation efficiency. A low level of nucleotide preference was observed at the junction sites using different synthetic DNAs. Furthermore, we discovered that T4 DNA ligase efficiently ligated DNA to the 3’ recessed end of RNA, not to that of DNA, in a DNA/RNA hybrid, whereas RNA ligases are less efficient in this reaction. These novel properties of T4 DNA ligase can be utilized as a broad molecular technique in many important applications. We performed a proof-of-concept study of a new directional tagmentation protocol for next generation sequencing (NGS) library construction that eliminates inverted adapters and allows sample barcode insertion adjacent to genomic DNA. 3’BL after single transposon tagmentation can theoretically achieve 100% usable template, and our empirical data demonstrate that the new approach produced higher yield compared with traditional double transposon or Y transposon tagmentation. We further explore the potential use of 3’BL for preparing targeted RNA NGS libraries with mitigated structure-based bias and adapter dimer problems.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1093/dnares/dsy037

Cites

26 of 26 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 3′ Branch ligation by T4 DNA ligase at non-conventional DNA ends formed by nicks, gaps, and overhangs. (a) Schematic representation of ligation assay with different DNA accepter types. The blunt-end DNA donor (blue) is a synthetic, partially dsDNA molecule with dideoxy 3′-termini (filled circles) to prevent DNA donor self-ligation. The long arm of the donor is 5′-phosporylated. The DNA acceptors were assembled using 2 or 3 oligos (black, red, and orange lines) to form a nick (without phosphates), a gap (1 or 8 nt), or a 36-nt 3′-recessive end. All strands of the substrates are unphosphorylated, and the scaffold strand is 3′ dideoxy protected. (b) Analysis of the size shift of ligated products of substrates 1, 2, 3, and 4, respectively, using a 6% denaturing polyacrylamide gel. Reactions were performed according to the optimized condition. The negative no-ligase controls (lanes 1, 3, 4, 6, 7, 9, 10, 12, and 13) were loaded at 1 or 0.5× volume of corresponding experimental assays. If ligation occurs, the substrate size is shifted up by 22 nt. Red arrowheads correspond to the substrate, and purple arrowheads correspond to donor-ligated substrates. Donor and substrate sequences in Supplementary Table S1. (c) Expected sizes of substrate and ligation product and approximate ligation efficiency in each experimental group. The intensity of each band was estimated using ImageJ and normalized by its expected size. Ligation efficiency was estimated by dividing the normalized intensity of ligated products by the normalized total intensity of ligated and unligated products.
Figure 1 3′ Branch ligation by T4 DNA ligase at non-conventional DNA ends formed by nicks, gaps, and overhangs. (a) Schematic representation of ligation assay with different DNA accepter types. The blunt-end DNA donor (blue) is a synthetic, partially dsDNA molecule with dideoxy 3′-termini (filled circles) to prevent DNA donor self-ligation. The long arm of the donor is 5′-phosporylated. The DNA acceptors were assembled using 2 or 3 oligos (black, red, and orange lines) to form a nick (without phosphates), a gap (1 or 8 nt), or a 36-nt 3′-recessive end. All strands of the substrates are unphosphorylated, and the scaffold strand is 3′ dideoxy protected. (b) Analysis of the size shift of ligated products of substrates 1, 2, 3, and 4, respectively, using a 6% denaturing polyacrylamide gel. Reactions were performed according to the optimized condition. The negative no-ligase controls (lanes 1, 3, 4, 6, 7, 9, 10, 12, and 13) were loaded at 1 or 0.5× volume of corresponding experimental assays. If ligation occurs, the substrate size is shifted up by 22 nt. Red arrowheads correspond to the substrate, and purple arrowheads correspond to donor-ligated substrates. Donor and substrate sequences in Supplementary Table S1. (c) Expected sizes of substrate and ligation product and approximate ligation efficiency in each experimental group. The intensity of each band was estimated using ImageJ and normalized by its expected size. Ligation efficiency was estimated by dividing the normalized intensity of ligated products by the normalized total intensity of ligated and unligated products.CC BY-NC · PubMed Central
Figure 2 Gel analysis of size shift of ligated products using 6% TBE polyacrylamide gel. Red arrowheads correspond to the substrate, and purple arrowheads correspond to donor-ligated substrates: substrate 5 (nick) (a), substrate 6 (1-nt gap) (b), substrate 7 (2-nt gap) (c), substrate 8 (3-nt gap), (d) and substrate 9 (3′-recessive end) (e). Three DNA donors with different bases at the 5′-end of the ligation junction (T, A, or GA) were examined. Donor and substrate sequences are summarized in Supplementary Table S1. (f) Table of ligation efficiency calculated based on normalized band intensity using ImageJ.
Figure 2 Gel analysis of size shift of ligated products using 6% TBE polyacrylamide gel. Red arrowheads correspond to the substrate, and purple arrowheads correspond to donor-ligated substrates: substrate 5 (nick) (a), substrate 6 (1-nt gap) (b), substrate 7 (2-nt gap) (c), substrate 8 (3-nt gap), (d) and substrate 9 (3′-recessive end) (e). Three DNA donors with different bases at the 5′-end of the ligation junction (T, A, or GA) were examined. Donor and substrate sequences are summarized in Supplementary Table S1. (f) Table of ligation efficiency calculated based on normalized band intensity using ImageJ.CC BY-NC · PubMed Central
Figure 3 3′ Branch ligation at the 3′ end of RNA in DNA/RNA hybrid. Schematic representation of 3′-branch ligation on a DNA/RNA hybrid with a 20‐bp complimentary region. We tested whether blunt-end DNA donors would ligate to the 3′-recessive end of DNA and/or to the 3′-recessive end of RNA. DNA(ON-21) hybridizes with the RNA strand (a), whereas DNA(ON-23) cannot hybridize with the RNA strand (b). (c, d) Gel analysis of size shift of ligated products using 6% denaturing polyacrylamide gel. The red arrowheads correspond to the RNA substrate (29 nt), and the green arrowhead corresponds to DNA substrate (80 nt). The purple arrowhead corresponds to donor-ligated RNA substrates. If ligation occurs, the substrate size would shift up by 20 nt. (c) Lanes 1 and 2, experimental duplicates; lanes 7–10, no-ligase controls; 10% PEG was added with T4 DNA ligase. (d) Lane 1, no-ligase control; lanes 2, 3, and 8, T4 DNA ligase with 10% PEG; lanes 4, 5, and 9, T4 RNA ligase 1 with 20% DMSO; lanes 6, 7, and 10, T4 RNA ligase 2 with 20% DMSO.
Figure 3 3′ Branch ligation at the 3′ end of RNA in DNA/RNA hybrid. Schematic representation of 3′-branch ligation on a DNA/RNA hybrid with a 20‐bp complimentary region. We tested whether blunt-end DNA donors would ligate to the 3′-recessive end of DNA and/or to the 3′-recessive end of RNA. DNA(ON-21) hybridizes with the RNA strand (a), whereas DNA(ON-23) cannot hybridize with the RNA strand (b). (c, d) Gel analysis of size shift of ligated products using 6% denaturing polyacrylamide gel. The red arrowheads correspond to the RNA substrate (29 nt), and the green arrowhead corresponds to DNA substrate (80 nt). The purple arrowhead corresponds to donor-ligated RNA substrates. If ligation occurs, the substrate size would shift up by 20 nt. (c) Lanes 1 and 2, experimental duplicates; lanes 7–10, no-ligase controls; 10% PEG was added with T4 DNA ligase. (d) Lane 1, no-ligase control; lanes 2, 3, and 8, T4 DNA ligase with 10% PEG; lanes 4, 5, and 9, T4 RNA ligase 1 with 20% DMSO; lanes 6, 7, and 10, T4 RNA ligase 2 with 20% DMSO.CC BY-NC · PubMed Central
Figure 4 (a–c) Schematic representation of three transposon tagmentation methods followed by PCR amplification using Pr-A (blue arrow) and Pr-B (green arrow). Two-transposon method (a); one Y transposon tagmentation with 3′-gap filling (b); one-transposon method with adapter ligation at 3′-gap (c). (d) Graph of amplification signal after purification using pr-A or pr-A with pr-B after the various tagmentation and gap ligation conditions.
Figure 4 (a–c) Schematic representation of three transposon tagmentation methods followed by PCR amplification using Pr-A (blue arrow) and Pr-B (green arrow). Two-transposon method (a); one Y transposon tagmentation with 3′-gap filling (b); one-transposon method with adapter ligation at 3′-gap (c). (d) Graph of amplification signal after purification using pr-A or pr-A with pr-B after the various tagmentation and gap ligation conditions.CC BY-NC · PubMed Central
Figure 5 Base distribution bias of Tn5-gap ligation (a), two transposons (b), and regular TA ligation (c). Only the first 20 bases from each end of the ligation are presented; adenine, blue; cytosine, orange; guanine, grey; thymine, yellow; the average and standard deviation of five-independent libraries are presented.
Figure 5 Base distribution bias of Tn5-gap ligation (a), two transposons (b), and regular TA ligation (c). Only the first 20 bases from each end of the ligation are presented; adenine, blue; cytosine, orange; guanine, grey; thymine, yellow; the average and standard deviation of five-independent libraries are presented.CC BY-NC · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Research works printed but not linked: 1

  1. Goryshin, 2016, Oligonucleotide replacement for di-tagged and directional libraries

Cited by

See all 13

Metadata

Authors
Wang, L., Xi, Y., Zhang, W., Wang, W., Shen, H., Wang, X., Zhao, X., Alexeev, A., Peters, B. A., Albert, A., Xu, X., Ren, H., Wang, O., Kirkconnell, K., Perazich, H., Clark, S., Hurowitz, E., Chen, A., Xu, X., Drmanac, R., & Jiang, Y.
Affiliations
  • Advanced Genomics Technology Lab, Complete Genomics Inc., 2904 Orchard Pkwy, San Jose, California, USA
  • Institute of Biochemistry, BGI-Shenzhen, Shenzhen, China
ORCID
Jiang, Yuan
Venue
DNA Research
Publisher
Oxford University Press (OUP)
Published
2018-11-13
Volume
26
Issue
1
Pages
45-53
ISSN
  • 1340-2838
  • 1756-1663
DOI
10.1093/dnares/dsy037
PMC
PMC6379041
Cited by
13
References
26
Language
en
Licence
creativecommons.org/licenses/by-nc/4.0/ (vor, 1 d)
Funders
Shenzhen Peacock Plan (KQTD20150330171505310)

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review

Data sources

Bibliographic data: Semantic Scholar, OpenAlex, Crossref.

Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).

Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.