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Preparing Your DNAs for SNP Genotyping

By Patricia Taillon-Miller
October 20, 2004

DNA Handling

Your DNA samples are the single most important component of the genotyping assay.

• If the DNAs are of poor quality or the concentrations of the DNAs are variable then your genotyping results will be poor.
• Most genotyping methods available today begin with a PCR amplification step.
• The good news is that they require very little DNA usually about 2 - 3 ng per reaction.
• But a nice even level of PCR product is needed for high quality results.
• It is important that the concentration of your DNA is determined as accurately as possible.

Quantifying your DNA

Estimating DNA concentration on gels

• This is the least accurate method to determine DNA concentration.
• If you elect to use this method take the photo of your gel at the point at which your unknown and your standard bands are just about to disappear from the photo. This will give you the most accurate estimate for this method.
• This method can use a large volume of DNA, depending upon the spectrophotometer that you use.
• Be sure you are in the linear range of your spectrophotometer when you take your readings.

Pico Green (Molecular Probes, Eugene OR)

• This is the most sensitive method for determining DNA concentration
• It uses the least amount of DNA
• It depends on the sensitivity of your detection equipment but for our equipment you only need about 10 µl of a 50 pg/µl solution.
• This is about a 10-4 dilution of most DNA preps from blood.

Arraying Your DNAs

• I recommend standardizing your DNA samples to a single concentration. Then when you array or dilute your DNAs you do not need to use a different volume for each sample.
• I also recommend making a MASTER PLATE of DNAs arrayed in a 96 well format that is approximately 50-100X more concentrated than the working concentration for your genotyping application.
• This will allow you to generate 96-well working plates without having to go back to your original stocks of tubes.
• Going back to the stocks of tubes is time consuming and can cause errors that were not on earlier versions of the working plates.
• Store your original tube stocks of DNA in a different location than your master plates. This will give you a back up in case of a disaster.
• Having a MASTER PLATE will also give you flexibility if you want to change protocols.
• I further recommend making enough MASTER PLATES to complete the entire project.
• It is important that you test each MASTER PLATE with a control assay. To do this, dilute a small amount to the working concentration and run a test genotyping assay on the samples. Use a very robust assay. It will verify that everything is working great at the start and is something that can be repeated in the future if you run in to problems. Save the data for future comparisons.
• DNA WORKING PLATES should be diluted to the working concentration from the MASTER PLATES.
• For FP-TDI the working concentration is 0.8ng/µl for high quality human DNAs such as those from Coriell Institute. We then use 3µl per well or 2.4ng/rxn. For lesser quality DNAs more may be needed. This can be determined empirically by looking at PCR success rates. Keep in mind that too much DNA can inhibit PCR so a lot more is not always better. Once the PCR looks good, a test of genotyping can be done using the control markers.
• Also the amount of DNA needed is determined by the copy number of your target sequence so the DNA concentration will need to be adjusted for smaller genomes.
• Each batch of DNA WORKING PLATES can be tested against control assays if desired. This may be important if there are changes or a long time has lapsed since using the DNA.
• Working DNA trays can be stored at -20C. Masters should be stored at -80C.
• It is a good idea to give your DNA batch numbers; it may help track down a problem in the future.
• Following the above recommendations should ensure consistent DNA quality.
• We array our DNA in 96 well 500µl deep-well trays. We get them from Midwest Scientific (P9606, $115). We do not require that you use these trays but we do charge for any changes that need to be made to the protocols to accommodate other plastics.
• To set up the PCR reactions we distribute 3 µl of working concentration DNA to each well of a 384-well tray. We spin down the plate and let the DNA air-dry overnight. These plates are stored desiccated and are good for months. For large projects (~125- 384 wells of genotyping per week) we make them in batches that will last a week. This is adjusted depending on the project and storage capacity.
• For FP-TDI our informatics allows 3 configurations on a 384 well plate: 4 assays X 96 DNAs, 2 assays X 192 DNA's, or 1 assay X 384 DNA's. All of our charges are based on the number of 384 well plates that are needed to complete your project so we recommend that you fill the space with samples to optimize your costs. For each SNP there must be a blank on the plate, i.e. a no-DNA control. Another useful control is to provide a duplicate of at least one sample.