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Fig. 3 | BMC Molecular Biology

Fig. 3

From: Coincidence cloning recovery of Brucella melitensis RNA from goat tissues: advancing the in vivo analysis of pathogen gene expression in brucellosis

Fig. 3

Overview of coincidence cloning procedure. Methodology is based on Azhikina et al. [28]. RNA extracted from SMLN tissues from each goat was used to generate total cDNA containing both host (blue) and bacterial (dark green) cDNA. Total cDNA and B. melitensis 16M total genomic DNA are fragmented using a restriction enzyme and the ends ligated with different suppressive adaptors (bacterial, host cDNA: yellow-blue; bacterial genomic cDNA: yellow-pink). Each cDNA sample is mixed with digested genomic DNA, and the mixture denatured and renatured in the presence of excess gDNA, generating both gDNA/gDNA homodimers and gDNA/cDNA heterodimers of complementary strands. The samples then undergo a two-step PCR amplification, resulting in selective enrichment of bacterial cDNA fragments. Libraries were prepared from amplified products and sequenced to obtain profiles of B. melitensis 16M gene expression within the tissue of infected hosts

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