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PABIO 551 Lecture 3

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PCR primers. Avoid hairpins, homology at 3' ends, repetitive ... Arbitrary primer PCR. Gene fusion by PCR. Intermediate products. I II III. PCRs: Final product ... – PowerPoint PPT presentation

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Title: PABIO 551 Lecture 3


1
PABIO 551 Lecture 3
  • Recombinant DNA Methods

2
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.
3
Restriction enzymes cut DNA molecules at specific
sequences
4
Protecting Native DNA from Restriction
5
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
6
Visualization of restriction fragments separated
by gel electrophoresis
7
Pulsed-field gel electrophoresis separates large
DNA molecules
8
Restriction site mapping
9
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

10
The polymerase chain reaction
11
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…..
12
PCR sensitivity
13
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

14
Nested PCR
30 cycles
30 cycles
Increase sensitivity
15
PCR of unknown sequence modified inverse PCR
Arbitrary primer PCR
16
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
17
Alternative gene inactivation ? Red Recombinase
Figure 1, Datsenko and Wanner, PNAS, 2000
18
Cloning Vectors
Vector Holds Plasmid lt15kb Phage (M13
lambda) 5 - 30 kb Cosmids 20-60
kb BACs 50-100s kb
19
Plasmids are extrachromosomal self-replicating
DNAs
Copy number (1 - 100s) is a property of the
plasmid
20
Cloning with plasmid vectors
21
Making a plasmid DNA library
22
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)

23
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
24
Labeling probes
  • Incorporate labeled nucleotides using Klenow
    fragment or Taq polymerase
  • Cy3 or Cy5
  • Digoxygenin

25
Oligonucleotide probes are designed based on
partial protein sequences
26
Southern blotting detects specific DNA fragments
27
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.
28
DNA sequencing the Sanger method
Four separate polymerization reactions are
performed
29
Sanger Method Dideoxyribonucleosides
Lodish, Fig 7-28
30
DNA sequencing the Sanger (dideoxy) method
31
Sequencing today
Still the Sanger method, but……………. Automated DNA
sequencing using fluorescent dyes and PCR.
32
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

33
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.

34
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

35
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
36
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
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