Transposon site hybridization in Mycobacterium tuberculosis.
Microarray mapping of transposon insertions can be used to quantify the relative abundance of different transposon mutants within a complex pool after exposure to selective pressure. The transposon site hybridization (TraSH) method applies this strategy to the study of Mycobacterium tuberculosis and can be adapted to the study of other microorganisms. This chapter describes the methods used to mutagenize mycobacteria with transposons, extract genomic DNA, amplify genomic DNA adjacent to transposon ends using polymerase chain reaction and T7 transcription, and synthesize labeled cDNA. It also describes methods used to construct an appropriate microarray, hybridize labeled cDNA, and analyze the microarray data. Important considerations involved in the experimental design of the selective pressure, the design of the microarray, and the statistical analysis of collected data are discussed.
Murry
Jeffrey P
JP
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA.
Sassetti
Christopher M
CM
Lane
James M
JM
Xie
Zhifang
Z
Rubin
Eric J
EJ
D004251:DNA Transposable Elements; D016680:Genome, Bacterial; D016254:Mutagenesis, Insertional; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000254:growth & development; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods*
D004251:DNA Transposable Elements