Il'jI Methods Useful in Genetics and Oncology
Results
For SYBR Green I expuiments, typical melting curves were obtained with a product Tm of 86.7°C (Fig. l). Very few primer-dimers were present, even with the lowest concentration of standard DNA, negative control or test DNA. We verified the
absence of non-specific PCR products and primer-dimers on a 2% agarose gel.
A classic calibration curve was obtained with a twofold dilution series of DNA.
Data were analyzed using the fit points method (2-points). Standard curves
obtained with SYBR Green I (Fig. 2) and hybridization probes (Fig. 3) were linear
over at least 4 orders of magnitude. When needed, concentrations of genomic
DNA were adjusted by appropriate dilution to enter the standard range. Genomic
DNA was then quantified by comparison to the standard concentration curve.
Comments
The purpose of this work is to quantify human genomic DNA by real-time PCR
using an external DNA concentration standard. Clearly, gene quantification normalized to the amount of DNA prepared from a certain volume of blood is inadequate, even when precision and reproducibility of the technique are emphasized.
Although the concentration of the genomic DNA standard may not be accurate, if
all samples are compared to a single standard, the relative values for all samples
will be correct. New standards should be compared to known standards (Fig. 4).
DNA quantification was performed on ethidium bromide stained gel and compared to DNA quantification with LightCycler hybridization probes. Although
similar values were obtained with both techniques, the precision of the LightCycler data was much better than gel quantification (Fig.S).
The LightCycler technique makes it possible to quantify small amounts of
genomic DNA (0.04-400 ng) precisely. Absorbance and ethidium bromide techniques are restricted to high DNA concentrations and provide approximate estimation of DNA concentration.
Application
Amplification of Retinoic X Receptor B gene with the LightCycler provides specific and precise quantification of human DNA and can be applied to any kind of
human DNA sample. Quantification of other genes is then related to a precise
DNA concentration or a precise number of genome copies.
We are currently quantifying low copy viral genes in human lymphocytes.
Therefore, we need accurate quantification of lymphocyte DNA. We perform such
quantification using RXRB amplification. We then perform viral DNA amplification from a well known quantity of lymphocyte DNA. Coamplification of RXRB
and the gene of interest in a single tube should give even more accurate quantification. In a recent article, Jabs et al. described such coamplification of the genomic C-reactive protein gene and Epstein Barr virus [3].
Results
For SYBR Green I expuiments, typical melting curves were obtained with a product Tm of 86.7°C (Fig. l). Very few primer-dimers were present, even with the lowest concentration of standard DNA, negative control or test DNA. We verified the
absence of non-specific PCR products and primer-dimers on a 2% agarose gel.
A classic calibration curve was obtained with a twofold dilution series of DNA.
Data were analyzed using the fit points method (2-points). Standard curves
obtained with SYBR Green I (Fig. 2) and hybridization probes (Fig. 3) were linear
over at least 4 orders of magnitude. When needed, concentrations of genomic
DNA were adjusted by appropriate dilution to enter the standard range. Genomic
DNA was then quantified by comparison to the standard concentration curve.
Comments
The purpose of this work is to quantify human genomic DNA by real-time PCR
using an external DNA concentration standard. Clearly, gene quantification normalized to the amount of DNA prepared from a certain volume of blood is inadequate, even when precision and reproducibility of the technique are emphasized.
Although the concentration of the genomic DNA standard may not be accurate, if
all samples are compared to a single standard, the relative values for all samples
will be correct. New standards should be compared to known standards (Fig. 4).
DNA quantification was performed on ethidium bromide stained gel and compared to DNA quantification with LightCycler hybridization probes. Although
similar values were obtained with both techniques, the precision of the LightCycler data was much better than gel quantification (Fig.S).
The LightCycler technique makes it possible to quantify small amounts of
genomic DNA (0.04-400 ng) precisely. Absorbance and ethidium bromide techniques are restricted to high DNA concentrations and provide approximate estimation of DNA concentration.
Application
Amplification of Retinoic X Receptor B gene with the LightCycler provides specific and precise quantification of human DNA and can be applied to any kind of
human DNA sample. Quantification of other genes is then related to a precise
DNA concentration or a precise number of genome copies.
We are currently quantifying low copy viral genes in human lymphocytes.
Therefore, we need accurate quantification of lymphocyte DNA. We perform such
quantification using RXRB amplification. We then perform viral DNA amplification from a well known quantity of lymphocyte DNA. Coamplification of RXRB
and the gene of interest in a single tube should give even more accurate quantification. In a recent article, Jabs et al. described such coamplification of the genomic C-reactive protein gene and Epstein Barr virus [3].