Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR

BMC Microbiology
Shaobin Zhong, Antony M Dean

Abstract

Insertion sequences (IS) are small DNA segments capable of transposing within and between prokaryotic genomes, often causing insertional mutations and chromosomal rearrangements. Although several methods are available for locating ISs in microbial genomes, they are either labor-intensive or inefficient. Here, we use vectorette PCR to identify and map the genomic positions of the eight insertion sequences (IS1, 2, 3, 4, 5, 30, 150, and 186) found in E. coli strain CGSC6300, a close relative of MG1655 whose genome has been sequenced. Genomic DNA from strain CGSC6300 was digested with a four-base cutter Rsa I and the resulting restriction fragments ligated onto vectorette units. Using IS-specific primers directed outward from the extreme ends of each IS and a vectorette primer, flanking DNA fragments were amplified from all but one of the 37 IS elements identified in the genomic sequence of MG1655. Purification and sequencing of the PCR products confirmed that they are IS-associated flanking DNA fragments corresponding to the known IS locations in the MG1655 genome. Seven additional insertions were found in strain CGSC6300 indicating that very closely related isolates of the same laboratory strain (the K12 isolate) may differ in t...Continue Reading

References

Aug 1, 1990·Bio/technology·H OchmanD L Hartl
Sep 1, 1991·Journal of Molecular Evolution·J G LawrenceH Ochman
Aug 1, 1991·PCR Methods and Applications·C Arnold, I J Hodgson
Aug 1, 1986·Theoretical Population Biology·S Sawyer, D Hartl
Jul 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·L GreenD L Hartl
Jan 1, 1983·Molecular & General Genetics : MGG·K NymanE Ohtsubo
Jun 11, 1996·Proceedings of the National Academy of Sciences of the United States of America·L DiatchenkoP D Siebert
May 20, 1997·Molecular & General Genetics : MGG·L MaoM D Gale
Sep 5, 1997·Science·F R BlattnerY Shao
Mar 31, 1999·Proceedings of the National Academy of Sciences of the United States of America·D PapadopoulosM Blot
Jan 10, 2001·Proceedings of the National Academy of Sciences of the United States of America·M M RiehleA D Long
Mar 22, 2001·DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes·T HayashiH Shinagawa
May 10, 2003·Proceedings. Biological Sciences·Lucinda Notley-McRobbThomas Ferenci
Jun 13, 2006·DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes·Yoshitoshi OguraTetsuya Hayashi
Jul 1, 1995·TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik·R C JansenC Dean

❮ Previous
Next ❯

Citations

Dec 7, 2013·Biology Letters·Ana SousaIsabel Gordo

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
electrophoresis

Software Mentioned

BLAST

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.