Detection of base analogs incorporated during DNA replication by nanopore sequencing.

Abstract:

:DNA synthesis is a fundamental requirement for cell proliferation and DNA repair, but no single method can identify the location, direction and speed of replication forks with high resolution. Mammalian cells have the ability to incorporate thymidine analogs along with the natural A, T, G and C bases during DNA synthesis, which allows for labeling of replicating or repaired DNA. Here, we demonstrate the use of the Oxford Nanopore Technologies MinION to detect 11 different thymidine analogs including CldU, BrdU, IdU as well as EdU alone or coupled to Biotin and other bulky adducts in synthetic DNA templates. We also show that the large adduct Biotin can be distinguished from the smaller analog IdU, which opens the possibility of using analog combinations to identify the location and direction of DNA synthesis. Furthermore, we detect IdU label on single DNA molecules in the genome of mouse pluripotent stem cells and using CRISPR/Cas9-mediated enrichment, determine replication rates using newly synthesized DNA strands in human mitochondrial DNA. We conclude that this novel method, termed Replipore sequencing, has the potential for on target examination of DNA replication in a wide range of biological contexts.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Georgieva D,Liu Q,Wang K,Egli D

doi

10.1093/nar/gkaa517

subject

Has Abstract

pub_date

2020-09-04 00:00:00

pages

e88

issue

15

eissn

0305-1048

issn

1362-4962

pii

5876287

journal_volume

48

pub_type

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