qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The overall goal of QPCR TAG analysis is to improve the throughput of the synthetic yeast genome project PCR TAG genotyping assay by implementing a real-time PCR based detection system and miniaturized reactions. This is accomplished by first using a bulk liquid dispenser to distribute QPCR master mix into each well of a 1536 multi-well plate. Next, a nanoscale acoustic droplet ejection robot transfers template DNA and primers into the wells of the multi-well plate.

Then the plate is sealed and the reactions are run through A-Q-P-C-R thermocycler. Finally, the realtime PCR data is analyzed to determine the presence or absence of synthetic or wild type PCR tags at each locus. Ultimately the QPCR tag workflow improves throughput.

The main advantage of this technique over the current PCR TAG assay is it allows us to circumvent the use of gel electrophoresis and manual annotation of those gels. We first had the idea for this method when we realized how time consuming and labor intensive PCR tagging on longer chromosomes was going to be. After culturing and harvesting approximately 2 million yeast cells add 200 microliters of yeast lysis, buffer, and pipette to resuspend.

The pellet add 300 microliters of acid washed glass beads to approximately one millimeter below the level of the cell, blurry. Then in a fume hood, add 200 microliters of phenol chloroform, isoamyl alcohol, invert the tube three times to mix and verify the cap is sealed. Then using a benchtop mixer, shake the tube for 10 minutes at room temperature after centrifuging transfer 75 microliters of the aqueous phase to a new micro fuge tube containing one milliliter of 100%ethanol.

Invert the tube 10 times to mix after spinning again, aspirate the supernatant without dislodging the pellet and use 500 microliters of 70%ethanol to wash the pellet. After removing the supernatant use 75 microliters of 10 millimolar triss pH 7.4 to resuspend the pellet and vortex to mix. After diluting the DNA one to 10 aliquot 30 microliters of diluted GDNA into the appropriate well of a source plate for nanoscale acoustic liquid dispensing.

Pre prepare 850 microliters of QPCR master mix in a 1.5 milliliter micro fuge tube and vortex to mix. Then centrifuge a solution at 20, 800 G and room temperature for two minutes to remove any bubbles. Then using a bulk dispenser, dispense 500 nanoliters per well into a 1536 multi-well plate.

Collect the QPCR master mix at the bottom of each well by briefly spinning the plate. Next, using the acoustic liquid transfer system and the details outlined in the text protocol, dispense five nanoliters of diluted GDNA and 10 nanoliters of 50 micromolar premixed forward and reverse primers into the desired wells of the multi-well plate. Acoustic droplet technology uses sound to vertically transfer small volumes of liquid from a source plate to an inverted destination plate.

Here template, DNA and primers are transferred in multiples of 2.5 nanoliter droplets into the wells of a 1536 multi-well plate. In order to set up the QPCR tag reactions. Next, use an optically clear seal and a microplate sealer system to immediately seal the 1536 multi-well plate.

Immediately centrifuge the sealed plate at 2000 G at room temperature for three minutes to collect all reagents at the bottom of the well to carry out realtime PCR program, the QPCR instrument, according to the guidelines in the text protocol, and hit run once complete. Verify that all the wells passed the master control, which indicates QPCR master mix was detected in the well. Any well that fails represents a potential false negative data point.

To analyze the data, visually inspect it within the QPCR software and or export the data for processing offline from the instrument. In this experiment, wild type and synthetic chromosome three PCR tag primer pairs were tested with yeast GDNA extracted from four different strains. Two with wild type chromosome three and two that encode a synthetic chromosome.

Three as expected synthetic primers, generally amplified synthetic DNA and not wild type DNA. Similarly wild type primers produced amplicons exclusively from wild type DNA. The observance of a small number of false positives and negatives is expected, and possible sources of these errors are described in the accompanying text protocol.

Once mastered, this technique can be finished in about an hour start to finish. Thanks for watching and good luck with your experiments.