Copies of given region of dna in matter of hours

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Unformatted text preview: copies of given region of DNA in matter of hours Technique exploits specificity of primers Allows for selective replication of chosen regions Termed target DNA Large amounts of DNA can be produced from very small sample ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science THE THREE-STEP AMPLIFICATION CYCLE Starting with double-stranded DNA molecule the process involves a number of amplification cycles PCR requires three step amplification cycle Step 1: double-stranded DNA denatured by heating to 95C Step 2: primers anneal to complementary sequence of target DNA Temperature is lowered to approximately 50C Step 3: DNA synthesis occurs with heat-stable DNA polymerase Temperature is raised to the optimal temperature for Taq DNA polymerase, approximately 70C DNA is amplified exponentially ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science STEPS OF A SINGLE CYCLE OF PCR ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science GENERATING A DISCRETE-SIZED FRAGMENT ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science THE NUMEROUS APPLICATIONS OF GENETIC ENGINEERING In medicine Many useful proteins Gene therapy In agriculture Use of genetic engineering allows for the direct transfer of desirable traits to agriculturally important animals and plants ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) Different strains of a species may have subtle variations in nucleotide sequences that give rise to a slightly different assortment of restriction fragment sizes Genomic DNA is isolated and digested with a restriction enzyme that cuts infrequently; the resulting fragments are separated by gel electrophoresis and then stained with ethidium bromide Two isolates of the same species that have different RFLPs are considered different strains ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science DNA FINGERPRINTING FOR IDENTIFICATION OF MICROORGANISMS...
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