By Andrei L. Osterman, Svetlana Y. Gerdes
ISBN-10: 1588293785
ISBN-13: 9781588293787
ISBN-10: 1597453218
ISBN-13: 9781597453219
This ebook features a accomplished choice of experimental and computational suggestions and strategies for microbial genome-scale essentiality stories, constructed and provided through the prime teams within the box. It comprises distinctive description of the tactics, dialogue of strength problems and screw ups. All protocols keep on with the winning tools in Molecular Biology™ sequence structure.
Read Online or Download Microbial Gene Essentiality: Protocols and Bioinformatics (Methods in Molecular Biology, Volume 416) PDF
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Extra info for Microbial Gene Essentiality: Protocols and Bioinformatics (Methods in Molecular Biology, Volume 416)
Sample text
Pooled kanamycin-resistant transformants are analyzed by PCR for genetic footprinting (19). 1. Amplification of Target DNA The in vitro transposition system allows high-density transposon mutagenesis of discrete subgenomic regions. For the genome analysis of H. influenzae essential genes, ~10-kb chromosomal regions, overlapping by ~5 kb and covering the entire genome (1830 bp), were systematically amplified by PCR. In these experiments, primers were designed using the MacVector sequence analysis program to have a 62°C theoretical melting temperature, 40% to 60% A+T composition, and absence of 3′ dimer formation.
Gerdes, personal communication). This latter approach has the advantage of ruling Knr 1 Genome Primer X’ Tn Primer 2 Knr X’ PCR products analyzed by PAGE 2 1 Fig. 9. Transposon footprinting. A large collection of transposon inserts are generated and pooled. The inserts in a general region are identified by performing a PCR reaction using two primers: a uniquely oriented transposon (Tn)-based primer and a primer corresponding with a given genomic site. All the transposon inserts in a given region can be identified by polyacrylamide gel electrophoresis (PAGE) of pooled PCR products.
Identification of pooled transposon insert locations through microarray analysis of transposon boundary transcripts. A transposon containing two outward-facing T7 promoters is used to generate a pool of inserts. The DNA from the transposition products is isolated, cleaved with a restriction enzyme, and used as templates for T7 RNA polymerase-catalyzed RNA synthesis. The location of the inserts is determined by microarray hybridization of the resulting RNA. Transposon-Based Strategies for Gene Essentiality Genes A B 23 C Top strand Array data Bottom strand Insert sites Fig.
Microbial Gene Essentiality: Protocols and Bioinformatics (Methods in Molecular Biology, Volume 416) by Andrei L. Osterman, Svetlana Y. Gerdes
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