1. Polymerase Chain Reaction

2. PCR Repetitive amplification of a piece or region of DNA Numerous uses
Straightforward amplification & cloning of DNA
Mutagenesis
Sequencing
RT-PCR – reverse transcription coupled with PCR to amplify mRNAs (cDNAs actually are template)
Production of cDNA libraries

3. PCR Requirements DNA template Oligodeoxynucleotide primers
DNA that will be amplified (copied)
Oligodeoxynucleotide primers
anneal to template to allow DNA replication
thermostable DNA polymerase
DNA polymerase extends the primers to synthesize a copy of the template DNA
thermostable polymerases allow automation and repeated rounds of DNA denaturation
deoxynucleotides and appropriate reaction conditions
dNTPs are incorporated into synthesized DNA, buffered pH, & Mg2+ to allow enzyme activity of DNA pol

4. PCR: The Process Begin with a DNA template Insert in vector
1st strand cDNA
Genomic DNA
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5. PCR: The Process
Denature template
Anneal primers
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6. PCR: The Process Extend primers with thermostable DNA polymerase Taq
Pfu
This ends a PCR cycle
Additional cycles will repeat these three steps
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7. PCR: The Process Beginning of 2nd cycle
Melt newly synthesized DNA from template
New strands of DNA are now also available as templates
Anneal primers
Extend primers
1 & 2 3

8. PCR: The Process Beginning of 3rd cycle
Melt newly synthesized DNA from template
All new strands of DNA are now also available as templates
Anneal primers
Extend primers

9. PCR: Yields How much amplification can be achieved?
Each cycle of PCR theoretically doubles the number of template molecules
Therefore the rate of amplification is 2n Where n is the number of amplification cycles
This will reach a practical maximum yield due to reagent (primer & dNTPs) concentration limits and maximum rate due to limiting enzyme concentrations. This upper limit is about 1x106 X amplification.

10. PCR: Yields
Example: Starting with 2ng of 5kb DNA template to amplify a 1Kb insert, what is the theoretical yield after 20 cycles? After 30?
How many template molecules are there?
= 5000bp X 660g bp/mol bp = 3.3x106 g template/mol template
= 2x10-9 g template  3.3x106 g temp/mol temp = 6x10-16 mol temp
= 6x10-16 mol temp X 6.02x1023 molec/mol = 3.64x108 molecules
2. How many molecules of insert can be made in 20 cycles? 30?
3.64x108 molecules x 220 = 3.8x1014 molecules – 106 X
3.64x108 molecules x 230 = 3.9x1017 molecules – 109 X

11. Using PCR to Map a Molecular Marker
Mapping
determining linkage distance between loci
measure % recombination
Molecular marker
a locus that does not necessarily encode a protein, yet has allelic variants
therefore, no affect on phenotype due to allelic variation will ever exist
however, this locus is linked to other loci and could be a useful tool in studying the linked loci
Allelic variation in the marker locus is called polymorphism
base changes - SNPs
sequence length – AFLPs, RFLPs

12. Marker we will map +/Y; b bibS/b bibS ♂ B/+; b bibS/+ bibL ♂ + + +
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B/+; b bibS/+ bibL ♂ +
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B/B ; + bibL/ + bibL ♀

13. Recombination in the F1 females
Parental chromosomes
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bib +
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Recombinant chromosomes +
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14. Mate with b bibS/b bibS male
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bib +
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bib P
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bib + R
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15. Analysis +P bP +F1 +F2 bF2 +F2 bF2 Parental Recombinant 750 500 400
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