Quantification of DNA by “Real Time PCR” Technique

For the amplification of DNA samples, the technique of quantitative PCR or “Real-Time PCR” was employed, using the Applied Biosystems 7500 Real-Time PCR system. Unlike a conventional PCR, where the amplified product, or amplicon, is detected after the reaction is finished by observing the relevant band of amplified DNA on an agarose gel, with the “Real-Time PCR” technique it is possible to measure the amplified DNA while the reaction is in progress. This allows one to determine the initial amount of DNA present in a sample much more accurately and with very high sensitivity. The quantization is performed by using a fluorescent probe [sonda] which is employed to obtain a measurement [valutazione] of the amount of PCR product present in each cycle of the reaction. In practice, the intensity of fluorescent signal measured during the exponential phase of the PCR reaction allows one to determine the amount of genetic material present at the beginning of the reaction. For the analysis of DNA samples, we used the “Quantifiler® Duo DNA Quantification Kit“, (product number 4387746, site number 1101024) product, also acquired from Applied Biosystems (Foster City, Ca, USA). This kit allows one to simultaneously identify the total amount of human and male human DNA in a sample. The results obtained by using this kit allow one to determine whether the sample contains a sufficient quantity to proceed with the STR (Short Tandem Repeat) analysis, and can be used to determine the relative amount of male and female DNA present in the sample under examination. Finally, the kit used in this analysis contains a specific internal control (IPC) that allows one to measure the presence of any inhibitors capable of compromising the PCR itself. The IPC is based on the use of a synthetic DNA sequence not present in nature. The “Quantifier Duo DNA Quantification Kit” is designed and optimized for usage with the Applied Biosystems 7500 Real-Time PCR system equipped with the SDS software, available at the Histology and Medical Embryology section of Sapienza University of Rome. The genes analyzed are the following: Ribonucleasis P RNA Component III (RPPH1), and the SRY gene, used respectively to amplify human and human male DNA.

 

Next: Preparation of Samples for Real-Time PCR