If primer dimer signals were avoided, and if there were no unspecific product amplification, the quantity of fluorescence detected should be proportional to the quantity of specific amplification product, giving the possibility of quantification.
Prior to amplification by PCR, the quantity of DNA of each sample was checked using a NanoDrop 2000 spectrophotometer (Thermo Scientific).
This often results in amplification failure when the quantity of starting material is limited.
Curves were generated by plotting the log 10 of the quantity of each template dilution against the C T value obtained during amplification of each dilution.
The real-time fluorescence values were measured by the quantity of a reporter dye FAM released during amplification.
The amplification of known quantities of spiked mammoth DNA were not inhibited by our DNA extracts in twenty independent qPCR reactions.
First, stochastic biases during the amplification of minute quantities of genomic DNA obtained from embryo biopsies (1 to 10 cells each) both limit and obscure the ascertainment of inherited alleles, while also introducing artifactual mutations [ 8- 10].
Yet, biallelic systems are associated with a low discrimination power which makes them sensitive to confounding factors such as transfusions associated to the surgery, inborn microchimerism [Rubocki et al., 2001] or other possible medically related contaminations linked to the amplification of minute quantities of DNA.
We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA.
Identification of contamination sources and prevention of contamination constitute a key strategic challenge when attempting PCR amplification of minute quantities of DNA, and can have a major impact on the quality and reliability of the data produced.
Moreover, when using AmpliTaq Gold (Applied Biosystems, CA, USA), we increased the number of amplification products in the negative controls when increasing the quantity of AmpliTaq Gold in the PCR while keeping the amplification efficiency at roughly the same level.
