DNA Techniques

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DNA Techniques

• Recombinant DNA
• DNA Sequencing
• Gel Electrophoresis
• DNA Amplification
• DNA Fingerprinting
• Gene Therapy

by no means all...
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Recombinant DNA

• Getting genes from one organism into another
• Target gene vs. vector
• Restriction endonucleases
• tool for cutting DNA prior to insertion

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Restriction Endonucleases

• Naturally occurring bacterial enzymes
• Cut DNA (of attacking viruses?)
• break phosphodiester bonds
• Named for organism of origin
• EcoRI came from E. coli strain RI

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Restriction Endonucleases

• Restriction restricted to cutting at very
  specific sites
• palindromic DNA sequences
• inverted repeats
• flush cutters and staggered cutters

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Sample of Restriction Enzymes and Cutting Sites
EcoRI 5G A A T T C C T T A A G 5
Staggered cut
EcoRII 5 G C C T G G C C G G A C C G 5
Staggered cut
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Staggered Cuts vs. Flush Cuts
5 G T Py Pu A C C A Pu Py T G 5
G T Py Pu A C C A Pu Py T G
"sticky ends"
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Sticky Ends

• Unpaired bases have affinity for other unpaired
  bases
• will reform hydrogen bonds w/ complementary bases

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--C G C T A C C A A G C T T A C C G A--
--G C G A T G G T T C G A A
T G G C T--
--G C T A C C A A G C T ----- A G C T T A C C G
-- --C G A T G G T T C G A ----- T C G A A T G G
C --
A G C T -------- -------- T C G A
Recombinant DNA
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Cloning

• Making multiple copies of a target gene
• Generally use bacteria as the factory
• reproduce incredibly quickly
• have natural VECTOR
• Vectors carry the target gene

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Cloning

• Vectors
• segments of DNA that carry target gene allow
  reproduction of gene
• plasmids
• phages
• cosmids
• part viral, part plasmid
• BACs - bacterial artificial chromosomes
• YACs - yeast artificial chromosomes

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Cloning
chromosome
Target gene
Need restriction enzyme site on either side of
target gene as well as w/in vector!
Vector
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Cloning

• Use SAME restriction endonuclease to cut out
  target gene and open vector
• Must use an enzyme that produces sticky ends!
• Will have complementary sticky ends on target and
  vector

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Cloning
Bacteria will now produce target gene product
Transform bacteria w/ vector
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Cloning

• Interferon was the first human protein to be
  cloned this way
• Human proteins produced w/ recombinant DNA
  technology
• insulin
• human growth hormone
• superoxide dismutase (heart attack)
• Factor VIII (hemophilia)

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• Human proteins produced w/ recombinant DNA
  technology
• Fertility hormones
• Interleukin-2 (kidney disease)
• erythropoietin (anemia)
• lung surfactant protein (premature infants)
• DNase (cystic fibrosis)
• Renin inhibitor (high blood pressure)
• colony stimulating factor (post chemo-therapy)

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Transgenic organisms

• Genes may be transferred from eukaryotic one
  organism to another
• plants that glow in the dark
• transferred luciferase gene
• plants w/ herbicide resistance
• cows w/ human lactoferrin gene
• binds iron, prevents infection, (infant formula)
• goats w/ human tissue plasminogen activator
• breaks up blood clots

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Gel Electrophoresis

• Used to separate molecules
• Protein, RNA, DNA
• Separation based on size of molecules, charge,
  shape, etc.
• Necessary for Southern blots, DNA sequencing, DNA
  fingerprinting, RFLP analysis, many other
  applications

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Protein gels

• Used to identify genotypes
• Electrophoretic patterns inherited as alleles
• Electromorphs or allelomorphs
• A one protein
• a slightly different protein

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Protein gels
1 2 3
Lane 1 homozygote for slow allele Lane 2
heterozygote for both alleles Lane 3 homozygote
for fast allele
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RFLPs Restriction Fragment Length Polymorphisms

• Differences in restriction sites DNA from
  different sources cut into different sizes
• Flush or staggered cutters
• Quantify differences between species relatedness
• Identify source of tissue samples (forensics,
  etc.)
• Markers for genetic disease

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RFLPs
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RFLPs
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RFLPs
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RFLPs as markers
Normal b-globin gene
Mst II site
Sickle cell b-globin gene
no restriction site
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RFLPs as markers
SS Ss ss
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RFLPs as markers
Ind. A Ind. B
SS Ss ss
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Southern Blots

• Way to visualize specific DNA segments
• Transfers DNA from gel to nitrocellulose filter
• Stains specific DNA segment w/ radioactive,
  single-stranded DNA probe
• Northern blots RNA
• Western blots protein

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Southern Blot
If stain for non-specific DNA, see a smear
1. Heat to separate strands 2. Add radioactive
probe
3. Cool, probe binds to complementary areas
4. Place filter on X-ray film, develop, see
bands!
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DNA Fingerprinting

• Very specific form of RFLP analysis
• allows differentiation even if no differences in
  restriction sites
• Uses VNTRs
• variable number of tandem repeats
• short, repeated sequences ( 4 bases)
• GGACGGACGGACGGACCC TGCCTGCC TGCCTG
• non-coding, highly variable between individuals

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DNA Fingerprinting - Parentage tests
child
mother
Mr. A Mr. B Mr. C
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DNA Sequencing

• Sanger or Dideoxynucleotide method
• Uses base analogues that are chain terminators
• Depends on ability of electrophoresis to
  distinguish between DNA segments that differ in
  length by only one base

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DNA Sequencing

• Dideoxynucleotides
• sugar missing two oxygens
• oxygen on 3 carbon missing
• no new nucleotide may bind
• chain terminator
• Copy DNA template using ddnucleotides

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DNA Sequencing

• Run four DNA synthesis reactions
• one with 4 normal bases and some ddATP
• one with 4 normal bases and some ddTTP
• one with 4 normal bases and some ddCTP
• one with 4 normal bases and some ddGTP
• Run a gel each rxn in a lane (4 lanes)

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3---------T A C T A T G C C A G A 5
5 primer
ddATP rxn
5 primer A T G A T A
5 primer A T G A
(Sometimes the entire segment is replicated.
This happens in all the rxns and can be ignored.)
5 primer A
Three different segments produced 1 base, 4
bases, and 6 bases in length ignoring primer.
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3---------T A C T A T G C C A G A 5
5 primer
ddTTP rxn
5 primer A T G A T A C G G T C T
5 primer A T G A T A C G G T
5 primer A T G A T
5 primer A T
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3---------T A C T A T G C C A G A 5
ddATP A T G A T A A T G A A
ddTTP A T G A T A C G G T C T A T
G A T A C G G T A T G A T A T
ddGTP A T G A T A C G G A T G A T
A C G A T G
ddCTP A T G A T A C G G T C A T G
A T A C
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3---------T A C T A T G C C A G A 5
Read sequence
Run on a gel
T C T G G C A T A G T A 5
A T G A T A C G G T C T A T G A T
A C G G T C A T G A T A C G G T A
T G A T A C G G A T G A T A C G A
T G A T A C A T G A T A A T G A T A
T G A A T G A T A
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Polymerase Chain Reaction (PCR)

• Used to amplify DNA samples
• Small tissue sample --gt enough DNA to do
  fingerprinting, etc.
• Repeated cycles of DNA replication
• Uses Taq polymerase

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PCR
heat
Cool in presence of primer (DNA
oligonucleotide) nucleotides, polymerase
Repeat cycle many times
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