DNA Technology

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DNA Technology

• Chapter 20

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Plasmid Use

• Plasmids are good tools for DNA Technology
• Can be isolated from bacterial cells
• Often carry resistance genes
• Isolated genes of interest can be inserted into
  the plasmid
• How is this insertion done?
• Restriction endonucleases (enzymes)

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Restriction Enzymes

• Where were restriction enzymes first found?
• Bacterial cells
• They were used to protect bacteria from intruding
  phage DNA
• Bacterial DNA is modified to protect it from its
  own restriction enzymes
• Restriction enzymes often cut DNA leaving sticky
  ends

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• Restriction sites are the regions on the DNA that
  the restriction enzyme cuts.
• Why are restriction sites palindromes?

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How are restriction enzymes used in DNA
technology?
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Cloning Genes of Interest

• How can a biologist make large amounts of a gene
  and thereby produce lots of protein products?
• Clone the genes in recombinant plasmids

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Which method of bacterial genomic alteration is
exploited in this process?
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Genomic Libraries
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cDNA

• What is the problem with inserting a human gene
  into a bacterial plasmid?
• Introns are not spliced in prokaryotes
• How can this problem be solved?
• Reverse Transcription of mRNA

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Why is cDNA shorter than the original eukaryotic
DNA?
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Probes

• Radioactive probes can tag a specific gene
  sequence within a mass of DNA
• Probes are single stranded compliments to known
  sections of the DNA

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Gel electrophoresis

• How does gel electrophoresis work?
• Uses electric charge to separate molecules based
  on their size
• What charge does DNA have?
• Negative
• Which sized fragments will move furthest through
  the gel?
• Smallest ones

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Restriction Fragment Analysis

• Genetic markers are regions of DNA that vary from
  person to person
• Usually located on non-coding regions of the DNA
• Using restriction enzymes and gel
  electrophoresis, DNA of different individuals can
  be analyzed and compared
• Extract DNA and treat it with restriction enzymes

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How could you detect the differences between
these 2 alleles?
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Using RFLP Analysis to Detect Harmful Alleles

• Harmful, disease causing alleles usually have
  identical RFLPs within a family
• Once the known RFLPs for the normal and disease
  causing alleles are known family members can be
  tested using Southern Blot analysis

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Southern Blot
Load the gel with the DNA to be tested. Markers
serve as standards for determining sizes of DNA
fragments.
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• Separated DNAs are denatured while still in the
  agarose, by soaking the gel in a basic solution
• Single stranded DNAs are transferred to a nylon
  membrane by blotting

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• A Radioactively labeled probe is added to the
  nylon membrane
• The probe is either RNA or DNA that will
  compliment a specific bp sequence on the DNA
• After unbound probes have been washed away only
  bound probes remain on the blot

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VNTR

• A VNTR is variable numbered tandem repeat
• Tandem repeats are interspersed throughout the
  genome
• Different VNTRs can be detected using southern
  blot technique

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One VNTR is inherited from each parent

• Southern blot analysis usually shows 2 different
  bands one inherited from each parent
• How could an individual have one band for the
  VNTR?
• He/She inherited the same sized VNTR from each
  parent

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Three different alleles for this particular VNTR
What are the different genotypes for these 6
individuals?
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Frequency of VNTRs

• Frequency of allele pattern at a single VNTR has
  been established for specific sites within the
  genome
• What is the probability of matching a 5 locus DNA
  profile, where each locus is0.01, 0.02, 0.03,
  0.06 and 0.10?
• One in 27.8 million people will randomly match
  this profile
• OJs profile was of 24 different loci and he
  matched all 24!
• The odds were 1 in 10 billion

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Amplification of DNA

• What enzymes would be necessary for DNA
  amplification?
• DNA polymerase and ligase
• What else would be necessary for the process to
  work?
• Primers and nucleotides!
• How was the original DNA initially uncoiled and
  unwound?
• Heat
• Why did the heat cause difficulty with the
  procedure?

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Mapping the Entire Genome

• Gene linkage mapping uses recombination
  frequencies to construct linkage maps of
  chromosome
• Chromosome walking will identify sequential
  regions of the chromosome

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Chromo-some Walking
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• DNA Sequencing uses defective nucleotides to
  sequence the DNA.

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In Situ Hybridization

• Denatured DNA is placed on slide
• radioactive single stranded probe is used to
  identify complementary DNA
• Used to identify genes that different species
  have in common

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Microassays use in situ hybridization technique
to determine which genes are actively being
expressed in the tissue sample
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Other uses for the new technology.
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Gene Therapy
Stem cells are the best candidates for this
therapy
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• Pharmaceutical Products
• Forensics
• Environment
• Agriculture
• Paleontology

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Determining PaternityWhich child cannot belong
to this set of parents?
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Which lane represents the father?
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Rape InvestigationDid the suspect commit the
crime?
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Supplemental Lab 6A

• In this lab we will transform E. coli bacterial
  colonies with recombinant plasmid DNA
• It will be your job to distinguish between
  bacterial colonies that have been transformed
  with the recombinant plasmids

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Procedure for the Transformation

• Click here to find out more about the procedure

Predict results for your procedure!