WHODUNNIT?

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WHODUNNIT?
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DNA Technology- What is it?

• Making recombinant DNA (DNA in which genes from 2
  different sources are combined)
• Biotechnology-manipulation of genes of organisms
  for our benefit
• Genetic engineering-directly changing genes of an
  organism
• Examples
• Cloning
• Gel electrophoresis
• Gene therapy
• Test-tube babies
• Sequence DNA/genes
• Stem cell research

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What can we do with it?The Big Picture

• Diagnose diseases
• Cure diseases
• Produce better medicines/vaccines more
  efficiently
• Solve crimes
• Paternity tests
• Produce more nutritious foods
• Produce food in countries with little or no
  arable land
• More efficiently Dispose of waste

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Important Techniques/Tools Used in DNA Technology

• Restriction Enzymes
• DNA libraries
• Gel electrophoresis
• Bacterial transformation, plasmids
• cDNA
• Polymerase chain reaction (PCR)
• probing/DNA hybridization
• DNA sequencing
• Bioinformatics
• Southern blotting
• Microarray analysis
• RFLPs
• vectors

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A few words about bacterial genetics.

• Prokaryotes
• Nucleoid region
• Reproduce by binary fission-108 in 12 hrs!!
• Genome
• 1 DS circular chromosome
• Plasmids-smaller circles of DNA
• Self replicating
• Confer new characteristics
• R plasmids, F plasmids
• Can be genetically engineered
• Genetic recombination in bacteria
• Transformation-uptake of naked foreign DNA
• Transduction-viruses
• Conjugation using pilli

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Restriction Enzymes

• Used in almost areas of recombinant DNA
  technology
• Create DNA libraries- collections of bacteria
  each having a random fragment of DNA

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Restriction Enzymes/Endonucleases

• Enzymes able to cleave DNA at specific sequences
  4-8 bps in length
• Originally found in bacteria, used as a defense
  against viral infection
• Presently over 200 known REs, each recognizing a
  different DNA sequence

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• May make blunt (linear) cuts or staggered (zig
  zag) cuts
• Staggered cuts leave sticky complementary
  ends-this allows DNA from 2 sources to join
  together (recombinant DNA)

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• Not every sequence is found in every genome/DNA
  fragment
• Enzymes recognizing smaller sequences usually cut
  more often than enzymes recognizing larger
  sequences
• Since every organisms DNA is different, cutting 2
  or more samples w/ the same RE will yield
  different sizes numbers of fragments
• Cutting one DNA sample w/ the SAME RE will yield
  fragments which can be used to create a
  restriction map

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Gel Electrophoresis

• Uses
• Paternity testing
• Solving crimes
• Diagnosing diseases
• Finding a particular gene or DNA sequence in a
  DNA library
• Taxonomical studies

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The basics.http//gslc.genetics.utah.edu/units/
biotech/gel
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A few more points..

• Can be used to separate proteins, lipids, nucleic
  acids
• Gel is porous agarose (starch or cellulose) and
  consistency can vary
• Rate of movement is a function of fragment
  length/agarose consistency/time/ and voltage
• Gels/DNA have dyes added to increase visibility
  of fragments both during after electrophoresis

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Whodunnit?Crime Scenes
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Whos my Baby Daddy?Aka paternity cases
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Size DOES Matter!Determining the size (in bps)
of DNA fragments of unknown lengths

• Generate a STANDARD CURVE using known sample
• Compare distance migrated of unknown fragments to
  standard curve
• Estimate unknown fragment sizes