Hrincevich_CH_13_student_outline

Hrincevich_CH_13_student_outline - Biotechnology Chapter 13...

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Biotechnology Chapter 13
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Biotechnology
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Genetic Engineering
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Recombinant DNA
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Discovery of Restriction Enzymes
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Specificity of Cuts Restriction enzymes cut DNA at a specific sequence # of cuts made in DNA will depend on number of times the “target” sequence occurs. For example: 5’-ATT(down)AAT-3’ EcoRI The “down” symbol indicates where the cut is made
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5’-CGGGCGCATTAATCCCATTAATGCG-3’ 5’-CGGGCGC ATTAAT CCC ATTAAT GCG-3’ 5’-CGGGCGC ATT AAT CCC ATT AAT GCG-3’ Cut DNA with Eco RI restriction enzyme In linear DNA, 2 restriction sites present will cut DNA into 3 fragments
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Making Recombinant DNA 5’ 3’ G C T T A A A A T T C G G A A T T C C T T A A G 3’ 5’ one DNA fragment another DNA fragment 3’ 5’
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Making Recombinant DNA nick 5’ 3’ 3’ 5’ G A A T T C C T T A A G nick G A A T T C C T T A A G DNA ligase action
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Using Plasmids Plasmid is small circle of bacterial DNA Foreign DNA can be inserted into plasmid Forms recombinant plasmids Plasmid is a cloning vector Can deliver DNA into another cell
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Using Plasmids DNA fragments + enzymes recombinant plasmids host cells containing recombinant plasmids
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Amplifying DNA Fragments can be inserted into fast-growing microorganisms Polymerase chain reaction (PCR) is a technique used to make lots of copies of DNA from a small starting amount “Xerox machine” for DNA
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Polymerase Chain Reaction Sequence to be copied is heated PCR requires a small piece of DNA called a primer that is complementary to the gene sequences targeted for copying DNA polymerase uses free nucleotides to create complementary strands Double number of copies of DNA
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4 Steps of a PCR Cycle 1. Template strand separation 2. Binding of the primers The temperature is lowered to 50 C to allow the primer DNA segments to bind to the targeted gene sequences through hydrogen bonding Sample is heated to 90-95 C to cause the double standard template DNA to separate into single strands…
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4 Steps of a PCR Cycle (cont’d.) 3.
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This document was uploaded on 10/26/2011 for the course BIOL 1001 at LSU.

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Hrincevich_CH_13_student_outline - Biotechnology Chapter 13...

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