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Microbiology a clinical approach by tony srelkauskas

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Unformatted text preview: Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science APPLICATIONS OF GENETIC ENGINEERING Researching gene function and regulation Express recombinant protein in E. coli Regulation of gene expression can be studied by creating a gene fusion Joining the gene being studied and a reporter gene Reporter gene encodes observable trait such as fluorescence GFP, green fluorescent protein Trait makes it possible to determine changes in gene expression ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science DNA SEQUENCING Allows the determination of the exact sequence of nucleotide bases in DNA Knowing DNA sequence of particular cell helps identify genetic alterations Alterations that may result in disease Sickle cell anemia Due to single base-pair change in gene Cystic fibrosis Caused by three-base pair deletion ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science TECHNIQUES USED IN DNA SEQUENCING Dideoxy chain termination method In vitro DNA synthesis reaction requires: Single-stranded DNA template, from which a complementary copy is sythesized Primer that anneals to single-stranded template Dictates the site at which synthesis will initiate DNA polymerase, the enzyme that catalyzes DNA synthesis Each of the four deoxynucleotides (dATP, dGTP, dCTP, dTTP) If these were the only ingredients in the reaction buffer, full-length molecules complementary to the template DNA would by synthesized. Dideoxynucleotides (ddNTPs) Identical to their deoxynucleotide counterparts but lack 3’OH group required for the addition of subsequent nucleotides during DNA synthesis Incorporation causes chain termination Special gel electrophoresis used to separate DNA fragments by size ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science DNA SEQUENCING: PRIMER DNA being sequenced usually has been cloned into a vector. DNA polymerase can only add nucleotides to an existing fragment of DNA, thereby extending the fragment ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science DNA SEQUENCING: CHAIN TERMINATION BY A DIDEOXYNUCLEOTIDE ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science AUTOMATED DNA SEQUENCING Automated DNA sequencing Most automated systems use fluorescent dyes to detect newly synthesized DNA Gel electrophoresis used to separate fragments into colored bands Laser used to detect color differences Order of color reflects nucleotide sequence of the DNA ISBN: 978-0-8153-6514-3...
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Microbiology A Clinical Approach by Tony Srelkauskas ©...

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