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PMID: 8341597 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Determination of DNA replication kinetics in synchronized human cells using a PCR-based assay.

Nucleic acids research ·Vol. 21 ·No. 14 ·1993-07-11 ·Pages 3227-32

Sinnett D, Flint A, Lalande M

Abstract

Studies on the temporal order of DNA replication are difficult due to the lack of sensitivity of methods available for replication kinetic analysis. To overcome problems associated with the current techniques, we propose a PCR-based assay to determine the replication time of any single-copy DNA sequence in complex genomes. Human cells labeled with 5-bromodeoxyuridine (BrdU) were flow sorted, according to their DNA content, at different times after synchronous release from the G1/S phase boundary. The selective removal of newly-replicated BrdU-substituted DNA was achieved by UV light irradiation followed by S1 nuclease treatment. The timing of replication of selected DNA sequences (housekeeping, tissue-specific, and non-coding loci) was determined by polymerase chain reaction (PCR) amplification using appropriate primers. DNA sequences localized in inactive replication units allowed amplification whereas those that have replicated will not be amplified by PCR. Using this sensitive and quantitative assay the replication kinetic analysis of a number of different DNA sequences can be performed from a single sorting experiment.

MeSH Terms
Bromodeoxyuridine Cell Line Cells, Cultured DNA/radiation effects DNA Replication Flow Cytometry Humans Kinetics Polymerase Chain Reaction/methods S Phase Single-Strand Specific DNA and RNA Endonucleases Ultraviolet Rays
Chemicals
DNA Single-Strand Specific DNA and RNA Endonucleases Bromodeoxyuridine
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Sinnett D
Genetics Division, Children's Hospital, Boston, MA 02115.
Flint A
Lalande M
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27 references, click to expand
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/21.14.3227
Published
1993-07-11
Pages
3227-32
Language
English
Region
England
NLM ID
0411011
PMCID
PMC309759
Subset
IM
Grants
NHGRI NIH HHS · R55HG00177-01 · United States
External Links
PubMed source DOI: 10.1093/nar/21.14.3227 PMC Full Text View journal details
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