1. PCR provides a forensics tool for identifying colonies
Yeast Colony PCR
PCR provides a forensics tool for identifying colonies
Three strains look alike!
How can you identify the strains?
Geneticists like to verify their strains’ genotypes before experiments

2. Discovery of the polymerase chain reaction expanded the reach of molecular biologists
Photo by Yellowstone NPS
Key discovery:
DNA polymerases from Thermus aquaticus and other thermophiles are active at high temperatures and do not denature at temperatures that denature DNA helices
PCR can generate a billion copies of a DNA sequence of interest
Investigators can generate custom DNA sequences with PCR
PCR is also a discovery tool, since only partial sequence information is needed to design primers

3. What principles are used to design PCR primers?
What happens at each temperature in a PCR cycle?
How can PCR be used to identify MET genes that have been disrupted by the insertion of a KANR cassette?

4. Good primer design is CRITICAL to the success of PCR reactions
PCR allows investigators to amplify any stretch of DNA as long as they know enough about its sequence to design primers
Yfg
Target sequence
Primer 1
Primer 2
Primers are oligonucleotides (~ bp) that bind to one strand of the DNA
Primers serve as the start site for DNA polymerases, which extend the primers in a 5’ to 3’-direction
Two primers are needed, one for each strand of the DNA
Good primer design is CRITICAL to the success of PCR reactions

5. G C A T The higher the Tm, the more specific that priming will be
Primer melting temperature (Tm) is critical for PCR
DNA polymerases can’t bind DNA in the absence of a primer
Number of hydrogen bonds dictates the stability of a hybrid
Increases with GC composition (more H-bonds that AT pairs)
Increases with length of the primer-template hybrid
G C A T
Quick and dirty calculation of the melting temperature (Tm) for short primers:
Tm = 2*(number of AT base pairs) + 4*(number of GC base pairs)
The higher the Tm, the more specific that priming will be

6. What principles are used to design PCR primers?
What happens at each temperature in a PCR cycle?
How can PCR be used to identify MET genes that have been disrupted by the insertion of a KANR cassette?

7. Researchers use a thermocycler for PCR reactions
Programmed to bring the reaction blocks through a series of temperature changes
Temperatures typically cycle between:
94-95˚C - DNA denatures (single-stranded)
55˚C – Primers anneal with target DNA
72 ˚C – Polymerases extend primers, copying DNA

8. Template DNA must first be denatured
94 ˚C
Denaturation
55 ˚C
Annealing
Annealing: primers bind to denatured DNA

9. DNA polymerases extend primers bound to the template DNA
Cycle 1
DNA polymerases extend primers bound to the template DNA
72 ˚C
Extension
DNA polymerases are processive: they copy the DNA until they “fall off”
Copies of the original template will vary greatly in their lengths

10. Extension products of the desired length begin to appear
Cycle 2
Extension products of the desired length begin to appear

11. Products of the desired length double with each cycle
Copies of the parental DNA are also made in each cycle, but they are dwarfed by products of the correct size

12. What principles are used to design PCR primers?
What happens at each temperature in a PCR cycle?
How can PCR be used to identify MET genes that have been disrupted by the insertion of a KANR cassette?

13. Our strains were produced by the Saccharomyces Genome Deletion Project
Homologous recombination was used to replace every ORF in the genome with a kanamycin resistance cassette
Yeast chromosome
Yeast ORF
5'-flanking region
3'-flanking region
1. PCR was used to generate upstream and downstream flanking regions for each ORF
PCR fragment
KanR
2. A series of additional PCR reactions were used to combine the flanking regions with the kanamycin cassette

14. Yeast were transformed with individual kanamycin resistance cassettes
PCR fragment
Yeast chromosome
Yeast ORF
5'-flanking region
3'-flanking region
KanR
Yeast were transformed with individual kanamycin resistance cassettes
Homologous recombination replaced the ORF with a kanamycin cassette
5'-flanking region
3'-flanking region
KanR
Recombinant chromosome
Where else is the homologous recombination machinery used?

15. Primer pairs can be used to distinguish the native and recombinant chromosomes
Native chromosome
Yeast ORF
5'-flanking region
3'-flanking region
GSP Primer A
GSP Primer B
Gene-specific (GSP) primers A and B give a product from the native chromosome, but not the recombinant chromosome
5'-flanking region
3'-flanking region
KanR
Recombinant chromosome
GSP Primer A
KAN Primer B
Gene-specific (GSP) primer A and KAN primer B give a product from the recombinant chromosome, but not the native chromosome

16. in the next lab, you will analyze the PCR products on agarose gels
The sizes of the PCR products are compared to the predicted values to identify the colonies
Size (bp)
in the next lab, you will analyze the PCR products on agarose gels
Colony PCR of strain BY4742 with gene-specific primers for MET genes. Gene-specific primers A and B were used for each PCR reaction. PCR products were separated by electrophoresis on 1% agarose gels. The sizes of the molecular weight standards (lane 1) are shown on the left. Primers used for the reactions correspond to MET1 (lane 2), MET3 (lane 3), MET7 (lane 4) and MET8 (lane5).