Applications and Basic Technology. Recombinant DNA technology : set of techniques for recombining...
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Transcript of Applications and Basic Technology. Recombinant DNA technology : set of techniques for recombining...
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BIOTECHNOLOGYApplications and Basic Technology
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Recombinant DNA technology
Recombinant DNA technology: set of techniques for recombining genes from different sources and transferring into cells where it may be expressed
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Restriction Enzymes
Occur in bacteria where they protect against intruding DNA
Involves restriction; foreign DNA is cut into small segments
Cut at specific points on small segments called specific recognition sequences
Several hundred restriction enzymes & ~ 100 different specific recognition sequences
Cuts DNA in staggered manner, resulting in single-stranded ends, called sticky ends
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Sticky Ends v. Blunt Ends
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Creates restriction fragments
Used in lab to join DNA pieces from different sources
Temporary bonds held by weak H bonds
Can be made permanent by adding DNA ligase
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Gel Electrophoresis
used to separate either nucleic acids or proteins based upon molecular size, charge and other physical properties
used to identify segments by looking at banding pattern when cut with restriction enzymes
DNA fragments can be isolated, purefied, and then recovered from gel
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Electrical current is used to move charged particles at different rates
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RFLP Analysis (restriction fragment length polymorphisms)
gel electrophoresis of restriction fragments results in characteristic banding pattern
Each band corresponds to DNA restriction fragments of certain length
Different alleles of gene result in dissimilar banding patterns
Similar patterns result when noncoding segments of DNA are used as starting materials
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Fragments move different distances based on base pair length, size (how compact)
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Used to match DNA from different sources
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Recombinant DNA
Use restriction enzymes to alter DNA Incorporate DNA into new organism to
create a GMO (genetically modified organism)
Ex: Super Salmon, Flavr Savr Tomato Super Salmon ABC Special
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Polymerase Chain Reaction
Technique used to amplify (copy many times) DNA in vitro (inside the cell)
1. DNA is incubated with special primers & DNA polymerase
2. billions of copies of the DNA are produced in a few hours
3. PCR is highly specific; primers determine sequence to be amplified
4. only minute amounts of DNA are needed
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