PCR Forensics

Today’s Lab•There has been an outbreak of Salmonella poisoning in the Student Union cafeteria at Stanford University cafeteria. You have been asked to identify the source of the Salmonella. Possible contaminated foods are: bad salami, unwashed lettuce, contaminated hamburger meat.

•You extract DNA from the three potentially contaminated foods.

•You also have a positive control (DNA from sample known to contain salmonella) and a negative control (DNA from sample known to not contain salmonella).

•Your job is to figure out which foods are contaminated.

•You will use PCR to figure out which sample is contaminated.

The 3 Steps of PCR1. Melt: Heating double-stranded DNA causes the strands to separate.

(95C)

2. Anneal: Cool to bind the primers to single stranded DNA. (65C)

3. Extend: Let polymerase extend off the primer (72C)

4. Back to step 1. Repeat 20 to 40 times. (See video)

Ingredients

• DNA template - DNA you want to amplify from

• Primers - bind to DNA and replication begins at these primed regions. Two primers are created specifically for the area of interest.

• Buffer - provides salt and pH

• Deoxynucleotide-triphosphates (dNTPs) - Mixture of dATP, dGTP, dTTP, dCTP, building blocks of DNA

• Mg2+ - cofactor for Taq, also changes salt conditions for reaction.

• Taq Polymerase

How do we do so many cycles?

95 degree - melt

65 degrees - anneal primers

72 degree extension

20 - 40 x

Now we use a machine called the

thermocycler

Use Gel Electrophoresis to observe different fragment lengths.

1. Load fragmented DNA into well.

2. Run a current through the gel. Negative at top, positive at bottom. DNA is negatively charged.

3. Large fragments move slowly, small fragments move fast.

4. Stain DNA to visualize.

Lab Setup• Use 5 thin-walled PCR tubes

• To each tube, add 10uL DNA from one food source, positive control, or negative control

• Add 15uL primer mix (forward and reverse primer already mixed for you)

• Add 25uL PCR master mix (contains buffer, Mg++, dNTPs, Taq polymerase)

Running PCR reactionPut your tubes in the PCR thermocycler and

run with these conditions:

94oC 2 minutes (to melt DNA)

94oC 30 seconds 50oC 30 seconds x35 cycles to amplify72oC 30 seconds

72oC 7 minutes4oC hold

Running a Gel

Expected results

Salmonella genomic DNA in food sample

Primer 1

Primer 2

Amplified Salmonella DNA (511 bp)

Expected results

•Positive control: band

•Negative control: no band

•Food samples: A DNA band indicates presence of Salmonella DNA.