Parameters Influencing Recombination Rates in PCR: More Dirty Sex in the Hood

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Parameters Influencing Recombination Rates in
PCRMore Dirty Sex in the Hood

• Presented by Wayne Yu
• Supervised by Prof. Niles Lehman

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Presentation Outline

• Recombination
• Definition contemporary examples
• Recombination During PCR
• Pros and Cons of PCR-mediated Recombination
• Project Goals
• Testing Recombination Parameters Using
  Tetrahymena
• Methodology
• Parameters
• Determination of Pause Sites
• Summary
• Future Projects

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What is Recombination?

• General Definition
• Exchange of genetic information between two
  sources. (Lehman N (2003) J. Mol. Evol.
  56770-777)
• i.e., Genetic Shuffling
• Examples of Recombination in Cells
• Segregation recombination of entire chromosomes
• Crossing-over of portions of chromatids
• RecA-type mediated recombination for DNA repair

Recombination in PCR was observed in the 1980s
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Normal PCR Schematic Diagram
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Enhanced Recombination in PCR (StEP)
Zhao et al. (1998) Molecular evolution by
staggered extension process (StEP) in vitro
Recombination. Nature Biotechnol. 16, 258-261
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Location Number of Crosses Can Vary
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Pros and Cons of Recombination

• Pros
• Nature uses it to create more genetic diversity
  on which natural selection can act.
• Allow efficient testing of evolutionary theories
• Escape from mutation melt-down (Mullers Ratchet)
  via regeneration of the most fit sequence
• Explore effective ways of searching sequence
  space
• PCR mediated recombination allows in vitro
  evolution for proteins with enhanced traits.
  (Zhao
  et al. (1998) Nature Biotechnol. 16, 258-261)
• Cons
• Forensic science relies on high fidelity
• RNA virus research clones PCR amplified DNA for
  virus strain characterization
• (Meyerhans et al. (1990), Nucleic Acids
  Research, 18 1687-91)

Goal To find parameters that will vary amount of
recombination in the PCR!
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Methodology

• Restriction Fragment Length Polymorphism
• (with a little bit of elementary math)

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Tetrahymena rRNA Self-Splicing Intron
The PCR product is 421 base pair long
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Tetrahymena Recombination
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Note Not drawn to Scale
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Tetrahymena RFLP Analysis
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Calculating Recombination Frequency
Our Definition of Recombination Frequency

421 (P1) 366 (K2) 266 (K1) 155
(P2K1) 55 (P2K2)
Number of recombinants Total number of sequences

• RF

K1 K2 P1 K2 (P2 K1)

266mer 366mer 421mer 366mer 155mer

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Actual Gel
421 (P1) 366 (K2) 266 (K1) 155
(P2K1) 55 (P2K2)
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Advantages Over the Traditional Method

• Much Greater Sampling Size
• Traditional methods involve cloning the PCR
  product and sequencing individual colonies
• We digest about 25 of PCR product for each RFLP
  experiment. (assuming 10 of the theoretic yield
  from PCR, we analyze 0.2 trillion molecules)
• The ease of quantitation method allow more
  efficient investigation.
• Effect of a parameter on RF can be studied in
  one single experiment

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Getting Started

• Different PCR Machines (Ramp Speed)

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Testing Parameter Ramping Speed
Thermal Program G1-2 (Normal PCR)
MJ PTC
GeneMate
P2 P1 Ctrl
Epp
A/B
Y101
Slow warming step increase RF?
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Ramping Speed continued.. Slow down
warming speed
52C to 72C _at_ 0.5C/sec
72C to 92C _at_ 0.5C/sec
P2 P1 Ctrl
Slowing down the ramp speed does not affect RF
much. Instead, we should try other parameters
Y105
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PCR Recombination Parameters

• Extension Time
• Extension Temperature
• Magnesium ion Concentration
• Input Template Concentration

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PCR with Various Extension Time
Y133R 10 seconds
Y140RR 2min
Y146 15min
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Extension Time and Temperature Data
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RF as A Function of Extension Time
Recombination drops when the extension time is
increased.
Explanation? As extension time increases, Taq is
more likely to fully extend the growing
strand. Meyerhans et al. found RF drop from 5.4
to 2.1 when extension time raise from 30sec to
3min
Why need 15 minutes??
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Single Stranded DNA Likely to Have 2º Structure
Tetrahymena ribozyme has many stem-loop structures
ScrF I cutting site
ssDNA during PCR can fold into some form of 2º
structure
Hpa I cutting site
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How Does 2nd Structure Cause Recombination During
PCR?

• What factors affect 2º structure?
• Temperature
• Salt Concentration

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Parameter 2 Extension Temperature
Similar trends of extension time are observed at
83C as well as at 88C
Unclear as to why RF increase back up a bit
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Although higher temperature can help weaken the
2nd structure, it may also lower the ability of
the growing strand to anneal to the
template (Weak effect)
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Parameter 3 Mg2 Concentration
Time
1min
2min
5min
10min
Mg2 mM
1 2.5 4 1 2.5 4
1 2.5 4 1 2.5 4
Y166
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RF Trend with Various Mg2
Possible Explanations Lower magnesium conc.
makes Taq less efficient for catalyzing
polymerization Magnesium stabilizes the dsDNA and
prevent it from falling off from the
template (Weak effect)
Dilemma Whatever stabilizes duplex DNA would
stabilize 2nd structure
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Parameter 4 Template Conc.
Normal PCR conditions
1 2 3 4 5 control
RF drops with less input DNA due to primers
ability to out-compete with partially extended
strand for the template strand.
Y169
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Summary Overall Picture
Longer Extension Time
Higher Extension Temperature
Lower Templates Concentrations
Lower Magnesium Concentration
Thermo-stable pyrophosphatase
Addition of DMSO
Vent DNA polymerase
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Determination of Pause Sites
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A Fragment was Observed!
240mer

• Two possibilities
• PCR artifact
• Reflecting pause site location

30 sec
3 min
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Future Projects Relate 2º Structure to
Recombination

• Recombine Weddell Seals DQA Gene
• Recombine DNA Template of 16s rRNA
• Lost of Recombination Via Mutagensis

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Recombining DQA GenesRecombining 16S rRNA DNA

• DQA gene should have little or no 2nd
  structure, thus wont be expect to pause Taq and
  recombine

16S rRNA has 2nd structure, but weaker than
Tetrahymena, so we expect medium amount of
recombination
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Expected Trend of Recombination with DQA Gene
and 16S rRNA
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Use Dirty to Destroy Sex
Mutagenic PCR should be able to weaken the 2º
structure, thereby smoothing out the extension by
Taq.