Molecular Combing of Single DNA Molecules on the 10 Megabase Scale

Scientific Reports
Atanas KaykovP Nurse

Abstract

DNA combing allows the investigation of DNA replication on genomic single DNA molecules, but the lengths that can be analysed have been restricted to molecules of 200-500 kb. We have improved the DNA combing procedure so that DNA molecules can be analysed up to the length of entire chromosomes in fission yeast and up to 12 Mb fragments in human cells. Combing multi-Mb-scale DNA molecules revealed previously undetected origin clusters in fission yeast and shows that in human cells replication origins fire stochastically forming clusters of fired origins with an average size of 370 kb. We estimate that a single human cell forms around 3200 clusters at mid S-phase and fires approximately 100,000 origins to complete genome duplication. The procedure presented here will be adaptable to other organisms and experimental conditions.

References

Jan 1, 1991·Methods in Enzymology·S MorenoP Nurse
Sep 30, 1994·Science·A BensimonD Bensimon
Oct 23, 1997·Biophysical Journal·J F AllemandV Croquette
Jun 5, 1995·Physical Review Letters·D BensimonA Bensimon
Mar 12, 1999·Science·M GrandboisH E Gaub
Apr 14, 1999·Nature Structural Biology·M RiefH E Gaub
Mar 29, 2000·Biophysical Journal·H Clausen-SchaumannH E Gaub
Mar 30, 2001·The Journal of Biological Chemistry·K Marheineke, O Hyrien
Oct 6, 2001·Science·M K RaghuramanW L Fangman
Nov 20, 2001·Nucleic Acids Research·K Otobe, T Ohtani
May 2, 2002·Proceedings of the National Academy of Sciences of the United States of America·Z GuerouiB Berge
Jul 19, 2005·Molecular and Cellular Biology·Ronald Lebofsky, Aaron Bensimon
Oct 28, 2005·Molecular Biology of the Cell·Prasanta K PatelNicholas Rhind
Oct 21, 2006·The EMBO Journal·Christian HeichingerPaul Nurse
Nov 15, 2007·Journal of Molecular Biology·Daniel M CzajkowskyZhifeng Shao
Sep 2, 2010·Proceedings of the National Academy of Sciences of the United States of America·Eva PetermannThomas Helleday
Sep 24, 2010·Nature Reviews. Molecular Cell Biology·Marcel Méchali
Dec 15, 2010·Nanotechnology·Annegret BenkeWolfgang Pompe
Dec 22, 2010·The Journal of Cell Biology·Xin Quan Ge, J Julian Blow
May 15, 2012·Methods : a Companion to Methods in Enzymology·Julien N BiancoPhilippe Pasero
Apr 6, 2013·Journal of Molecular Biology·Hervé TécherBenoît Le Tallec
Oct 29, 2013·Methods in Molecular Biology·Attila Németh
Jul 7, 2014·Nature Structural & Molecular Biology·Roxana E GeorgescuMike E O'Donnell

❮ Previous
Next ❯

Citations

Jun 4, 2016·Molecular Cell·Juan Carlos Rivera-Mulia, David M Gilbert
Jan 18, 2017·Proceedings of the National Academy of Sciences of the United States of America·Tea TotevaGeneviève Thon
Nov 23, 2017·Nature Communications·Andrey MikheikinJason Reed
Aug 22, 2018·Current Protocols in Microbiology·Catherine J RedmondAlison A McBride
May 3, 2016·Nature Communications·Sandro MorganellaSerena Nik-Zainal
Mar 29, 2019·Nucleic Acids Research·Nathaniel O WandRobert K Neely
Mar 28, 2018·The EMBO Journal·Néstor García-RodríguezHelle D Ulrich
Nov 30, 2018·Nanotechnology·Shuyi WuYuval Ebenstein
Nov 21, 2018·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Anna Ligasová, Karel Koberna
Feb 22, 2018·PLoS Genetics·Anthony PerrotPei-Yun Jenny Wu
Jun 11, 2021·Langmuir : the ACS Journal of Surfaces and Colloids·Akihiro OhmatsuzawaJiro Kumaki

❮ Previous
Next ❯

Methods Mentioned

BETA
electrophoresis
DNA shearing
FACS

Software Mentioned

Excel

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.