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

Photo by Yellowstone NPS

Discovery of the polymerase chain reaction expanded the reach of molecular biologists

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

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?

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Target sequence

Primer 1

Primer 2

PCR allows investigators to amplify any stretch of DNA as long as they know enough about its sequence to design

primers

• Primers are oligonucleotides (~18-25 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

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

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?

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

Template DNA must first be denatured

94 ˚CDenaturation

55 ˚CAnnealing

Annealing: primers bind to denatured DNA

72 ˚CExtension

Cycle 1

DNA polymerases extend primers bound to the template DNA

DNA polymerases are processive: they copy the DNA until they “fall off”

Copies of the original template will vary greatly in their lengths

Cycle 2

Extension products of the desired length begin to appear

Cycle 3

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

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?

Yeast chromosome

Yeast ORF5'-flanking region 3'-flanking region

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

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

5'-flanking region 3'-flanking regionKanR

Recombinant chromosome

PCR fragment

Yeast chromosome

Yeast ORF5'-flanking region 3'-flanking region

KanR

3. Yeast were transformed with individual kanamycin resistance cassettes

Where else is the homologous recombination machinery used?

4. Homologous recombination replaced the ORF with a kanamycin cassette

Primer pairs can be used to distinguish the native and recombinant chromosomes

Native chromosome

Yeast ORF5'-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 regionKanR

Recombinant chromosomeGSP 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

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).

1 2 3 4 5

Size (bp)

The sizes of the PCR products are compared to the predicted values to identify the colonies

in the next lab, you will analyze the PCR products on agarose gels