PABIO 551 Lecture 3

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PABIO 551 Lecture 3

• Recombinant DNA Methods

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Cloning depends on restriction enzymes
Restriction/modification systems Exist to
restrict (cut and destroy) foreign DNA. Host
DNA is saved by methylation. Types I and III
one protein w/ restriction modification
activities. Type II restriction and methylation
activities are different proteins. Cut sites
show two-fold symmetry. Cuts can leave 5,
blunt, 3 overhangs. Hundreds have been purified
for use in cloning.
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Restriction enzymes cut DNA molecules at specific
sequences
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Protecting Native DNA from Restriction
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Frequency of restriction enzyme cuts
Probability (4 possible bases at each position)
4 base recognition cuts every 44 256 bases 6
base recognition cuts every 46 4096 bases 8
base recognition cuts every 48 65,536
bases Frequency distorted base composition of
genome methylation repetitive DNA coding regions
usually normal non-coding, AT rich - most
enzymes, GC rich
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Visualization of restriction fragments separated
by gel electrophoresis
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Pulsed-field gel electrophoresis separates large
DNA molecules
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Restriction site mapping
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Polymerase Chain Reaction

• Used to specifically amplify a region of DNA
• Molecular Xerox machine
• Components
• DNA template
• Oligonucleotide primers -- matched Tm
• Mg -- DNA pol co-factor, need right
• dNTPs
• Thermostable polymerase -- differ in fidelity,
  processivity
• Salt

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The polymerase chain reaction
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PCR primers
Avoid hairpins, homology at 3 ends, repetitive
regions Tm calculations 4G/C 2A/T, but
very dependent on salt. For a 20-mer 12 G/C and
8 A/T 12(4) 8(2) 64 81.5 - 16.6(logNa)
0.41(GC) - (600/N) Salt conc. Tm 0.01 52.9
C 0.05 64.5 C 0.10 69.5 C 1 x SSC 85.3
Use computer programs..
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PCR sensitivity
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Modifications of PCR

• Normal PCR
• Nested PCR
• Fusion PCR
• RT-PCR
• Q-PCR
• Q-RT-PCR

• RACE
• Inverse PCR
• Multiplex PCR
• Gene inactivation with lambda red recombinase

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Nested PCR
30 cycles
30 cycles
Increase sensitivity
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PCR of unknown sequence modified inverse PCR
Arbitrary primer PCR
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Gene fusion by PCR
PCRs
I II
III
A B C D
A
D
Intermediate products
Final product
Nucleic Acids Res. 1989 Jun 2617(12)4895
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Alternative gene inactivation ? Red Recombinase
Figure 1, Datsenko and Wanner, PNAS, 2000
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Cloning Vectors
Vector Holds Plasmid lt15kb Phage (M13
lambda) 5 - 30 kb Cosmids 20-60
kb BACs 50-100s kb
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Plasmids are extrachromosomal self-replicating
DNAs
Copy number (1 - 100s) is a property of the
plasmid
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Cloning with plasmid vectors
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Making a plasmid DNA library
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Screening libraries

• 1. Array your library (plates)
• 2. Transfer to a filter
• 3. Denature (if DNA)
• 4. Probe
• 5. Wash
• 6. Develop
• Probes cDNA, PCR product, oligonucleotides,
  antibodies
• Could also screen using reporter assay (B-gal,
  luciferase)

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Alternative cloning Gateway Cloning
X
attB1
ccdB toxin
Gene X
attB2
1. PCR
Gene X
attL1
2. BP Clonase Rxn
attL2
Entry Vector
ccdB toxin
attP1
attP2
Donor Vector
3. Transform into E. coli
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Labeling probes

• Incorporate labeled nucleotides using Klenow
  fragment or Taq polymerase
• Cy3 or Cy5
• Digoxygenin

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Oligonucleotide probes are designed based on
partial protein sequences
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Southern blotting detects specific DNA fragments
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How many to screen?
The Candida albicans genome is 2 x 107 bp 2 x
107 bp/ 5 x 103 bp (avg. plasmid insert) 4000 2
x 107 bp/ 2 x 104 bp (avg. lambda insert)
1000 2 x 107 bp/ 2 x 105 bp (avg. BAC insert)
100 Number of clones (N) to guarantee
probability (P) that a sequence is in your
library
ln(1-P) ln(1-INSERT
SIZE/GENOME SIZE)
N
For 99 probability, N 4602 for lambda.
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DNA sequencing the Sanger method
Four separate polymerization reactions are
performed
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Sanger Method Dideoxyribonucleosides
Lodish, Fig 7-28
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DNA sequencing the Sanger (dideoxy) method
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Sequencing today
Still the Sanger method, but. Automated DNA
sequencing using fluorescent dyes and PCR.
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What is a microarray?

• A glass slide where DNA sequences from each gene
  is represented
• PCR (300-500 bp)
• Oligonucleotides (40 to 80mers)
• (Affymetrix--multiple 25-mers/ORF, also includes
  probes for intergenic regions)
• Utilizes complementary base pairing of labeled
  probes (Cy3 or Cy5) to DNA sequences on
  microarray
• Use bioinformatics program to help sort through
  data

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Applications of DNA microarrays

• DNA/DNA hybridizations
• Investigate gene content between different
  strains
• Expression profiling
• Comparing expression levels of two conditions
• Identification of protein binding sites
• ChIP-Chip. Immunoprecipitation of protein/DNA
  complexes. Assay interactions with microarrays.

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Applications of DNA Microarrays

• Disease processes
• Immune response to infection
• Pathogen gene expression in different environs
• ID essential genes required for certain
  conditions
• Gene expression in chronic disease processes
• Gene Discovery
• Disease diagnosis
• Drug Effects

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Microarray experimental overview
Condition 1
Condition 2
Collect cells from two different conditions
Isolate RNA/DNA, Make labeled cDNA
Hybridize on to microarray
Wash, Scan slide, Analyze data
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Monitoring changes in yeast gene expression in
presence or absence of glucose
Green- increased expression
Red decreased expression
Yellow equal expression
DeRisi et al. Science1997278680