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Real Time PCR as a Method to Characterize DNA
Transnetyx, Inc.
Introduction
Our current genotyping method includes measuring the optical density of each sample after the DNA is isolated. The final step of the Transnetyx genotyping method is a real time PCR reaction. Each sample is tested with a probe or a group of probes that was designed specifically for the strain. Each sample is also run with a positive control house-keeping gene. The house-keeping gene is used to characterize the DNA quantity and quality as well as be used mathematically to determine a “relative copy number” or RCN.
Optical density measurements are a common method for quantifying DNA yield. This method is popular for several reasons. Optical density measurements are passive; they do not affect the sample or react with it in any way. As a result, a concentration can be determined without losing any sample yield. It is quick; with the correct equipment, 384 samples can be measured in less than a minute. However, there are some drawbacks to using optical density as a means to determine DNA concentration. The method does not distinguish between types of nucleic acids. Single stranded DNA, double stranded DNA and RNA are all detected with an OD. Also, an OD is not sensitive to contaminates in the sample.
Real time PCR can also be used to quantify DNA yield, however sample is consumed during the process. Also, preparing a sample for real time PCR is both timely and expensive. However, real time PCR can be more informative than optical density measurements. PCR is very susceptible to contamination. If a sample yields good amplification, it is very likely that there is little to no contamination in the sample. Also, both single stranded and double stranded DNA can be detected with real time PCR.
Purpose
We noticed through the course of testing, that samples that fall below the detectable limits of the optical density reading often have very good, and useful amplification results. As we began to look closely at OD data versus real time PCR data, we found that the OD data did not add any value to our process. Although OD readings are inexpensive and fast, our protocol already involves the downstream use of real time PCR.
Experimental Design
A serial dilution of whole genomic mouse DNA was performed, resulting in DNA samples ranging from 1.875 - 60 nanograms per microliter. The DNA was loaded into the wells of a 384 well optical plate and an OD260 measurement was taken on a Tecan Genios Instrument with the following settings:
Meas. Mode: Absorbance
Ref. Wavelength: 0 nm
Number of Flashes: 5
Temperature: 24.3C
Volume: 100μL
The DNA was then used as template in a Real Time PCR run using two different real time reactions. The first probe is the standard, Transnetyx, house-keeping probe that is run with every sample, the second is a standard wild type probe that we developed for one of our customers. Universal PCR conditions were used with 20mL reaction volumes. Each sample was run in triplicate for each probe.