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Title: Identification of transposon insertion sites using the T7 RNA polymerase promoter      
keywords:
Transcriptome or Gene expression
ID:
PRJNA98531
description:
Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput genome-wide technique, Genomic Array Footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in non-reproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library, showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1, showed that both have an equal degree of saturation, but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for survival of zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae. Keywords: GAF Identification of transposon insertion sites Overall design: Genomic DNA (> 10 µg) was digested overnight with TaqI, DdeI or AluI and the purified DNA was used for an in vitro transcription reaction with the MEGAscript T7 RNA polymerase kit (Ambion) in a total volume of 20 ul.
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landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA98531
authentication:
none
authorization:
none
dateReleased:
06-26-2007
name:
Streptococcus pneumoniae R6
ncbiID:
ncbitax:171101
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject