Heteroduplex analysis using flow cytometric microbead assays to detect deletions, insertions, and single-strand lesions

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
Eva HegedusGabor Szabo

Abstract

We explore the possibilities offered by flow cytometric microbead analysis to develop high throughput methods for the detection of deletions/insertions and single-strand DNA lesions. The products of PCR reactions derived from reference and test samples are denatured and reannealed, then exposed to enzymatic or chemical treatments distinguishing homoduplices from heteroduplices. The biotin- and dye labeled reaction products are immobilized on microbeads and the homo- and heteroduplices are assessed in separate fluorescence channels, by flow cytometry. Using a model system based on the mixed lineage leukemia gene breakpoint cluster region, we demonstrate that deletions and insertions in genomic DNA can be detected, using S1 nuclease and chemical cleavage to distinguish hetero- from homoduplices, or a restriction enzyme cleaving only the homoduplices. Single-strand discontinuities can also be detected, by combining nick-translation, using labeled nucleotide, and flow cytometric microbead analysis. The methodical approaches demonstrated are applicable in a versatile manner in basic cell and molecular biological research and also promise direct application for high throughput screening of genetic diseases and lesions, including inse...Continue Reading

References

Jun 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·R G CottonR D Campbell
Dec 28, 1981·Biochimica Et Biophysica Acta·J R Silber, L A Loeb
Apr 1, 1997·Proceedings of the National Academy of Sciences of the United States of America·L NarayananP M Glazer
May 26, 1998·Human Mutation·T P EllisR G Cotton
Feb 15, 2003·FEMS Microbiology Reviews·Neelam A Desai, Vepatu Shankar
May 15, 2003·BMC Chemical Biology·Chinh T BuiRichard GH Cotton
Mar 30, 2004·Clinical Immunology : the Official Journal of the Clinical Immunology Society·Edward MorganAmitabh Gaur
May 11, 2004·Genes & Development·Angelika Daser, Terence H Rabbitts
Jan 22, 2005·Trends in Genetics : TIG·Peter D Keightley, Brian Charlesworth
Feb 23, 2005·Expert Review of Molecular Diagnostics·Ene-Choo Tan, Poh San Lai
Sep 16, 2005·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Judit PatakiGábor Szabó
Oct 1, 2005·Histochemistry and Cell Biology·Lóránt SzékvölgyiGábor Szabó
Jan 24, 2006·Journal of Molecular Biology·Andriy KhobtaGiovanni Capranico
Aug 1, 1997·Cell Death and Differentiation·P R WalkerM Sikorska
Mar 15, 2006·The Journal of Biological Chemistry·Negar S RahmanKarla K Rodgers
Apr 11, 2006·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·John P Nolan, Francis Mandy
Feb 10, 2007·The EMBO Journal·Gulnara AbdurashidovaArturo Falaschi

❮ Previous
Next ❯

Citations

Jul 21, 2011·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·László ImreGábor Szabó

❮ Previous
Next ❯

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.

Related Papers

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
Laszlo ImreGabor Szabo
DTW. Deutsche tierärztliche Wochenschrift
H FuhrmannH P Sallmann
Proceedings of the National Academy of Sciences of the United States of America
Lóránt SzékvölgyiGábor Szabó
© 2021 Meta ULC. All rights reserved