Quantification of Human Genomic DNA
Using Retinoic X Receptor B Gene
NATHALIE PIERI-BALANDRAUD, JEAN ROUDIER, CHANTAL ROUDIER*
Introduction
The LightCycler** system performs real-time PCR with specific and quantitative
analysis of amplified products. All parameters of the PCR reaction have to be
carefully adjusted. The most important factors are DNA quality and concentration. Pure DNA is prepared with any manufactured kit. Quantification of genomic DNA samples is essential for accurate quantification of small differences in
gene copy numbers or low copy target genes. Classic methods used to quantify
genomic DNA have limitations.
The traditional method to determine the amount of DNA in solution by measuring ultraviolet absorbance at 260 nm is easy to perform but has serious limitations [1]; for example, absorbance readings cannot discriminate between DNA and
RNA. The absorbance ratio at 260 and 280 nm is used as an indicator of nucleic
acid purity. Proteins have a peak absorption at 280 nm and reduce the A260/ A280
ratio. Ratios of 1.8 to 2 indicate highly purified preparations of DNA or RNA.
Absorbances at 230 nm and 325 nm reflect contamination by phenol, urea and particulates, and dirty cuvettes, respectively. Absorbance at 320 nm should be approximately zero with pure samples. Therefore, careful handling of sample cuvettes is
recommended. A large amount of DNA is required to obtain accurate readings. An
absorbance of 1.0 at 260 nm indicates 50 Jlg/ml of double-stranded DNA.
Ethidium bromide fluorescent quantification of doubled-stranded DNA is also
widely used [1]. Electrophoresis through gels containing ethidium bromide
(0.5Jlg/ml) is carried out for this purpose. Known volumes of DNA samples and a
series of standard DNA solutions (500,250,125,63,31 and 15 ng/6 JlI) are loaded
on the same gel. After migration the gel is photographed using short-wavelength
ultraviolet radiation. The intensity of fluorescence of the unknown DNA is compared with that of the DNA standards and the quantity of DNA in the sample is
estimated. A critical parameter is the "visual reading" of fluorescence.
We developed a simple technique to quantify DNA using the LightCycler System. Retinoic X Receptor B is a human single-copy gene mapping to chromosome
* Chantal Roudier (~) (email: chantal.roudier@medecine.univ-mrs.fr)
Laboratoire Immunorhumatologie, INSERM EMI 9940, Faculte de Medecine,
27 bd Jean Moulin, 13005 Marseille, France
** LightCycier is a trademark of a member of the Roche Group
Using Retinoic X Receptor B Gene
NATHALIE PIERI-BALANDRAUD, JEAN ROUDIER, CHANTAL ROUDIER*
Introduction
The LightCycler** system performs real-time PCR with specific and quantitative
analysis of amplified products. All parameters of the PCR reaction have to be
carefully adjusted. The most important factors are DNA quality and concentration. Pure DNA is prepared with any manufactured kit. Quantification of genomic DNA samples is essential for accurate quantification of small differences in
gene copy numbers or low copy target genes. Classic methods used to quantify
genomic DNA have limitations.
The traditional method to determine the amount of DNA in solution by measuring ultraviolet absorbance at 260 nm is easy to perform but has serious limitations [1]; for example, absorbance readings cannot discriminate between DNA and
RNA. The absorbance ratio at 260 and 280 nm is used as an indicator of nucleic
acid purity. Proteins have a peak absorption at 280 nm and reduce the A260/ A280
ratio. Ratios of 1.8 to 2 indicate highly purified preparations of DNA or RNA.
Absorbances at 230 nm and 325 nm reflect contamination by phenol, urea and particulates, and dirty cuvettes, respectively. Absorbance at 320 nm should be approximately zero with pure samples. Therefore, careful handling of sample cuvettes is
recommended. A large amount of DNA is required to obtain accurate readings. An
absorbance of 1.0 at 260 nm indicates 50 Jlg/ml of double-stranded DNA.
Ethidium bromide fluorescent quantification of doubled-stranded DNA is also
widely used [1]. Electrophoresis through gels containing ethidium bromide
(0.5Jlg/ml) is carried out for this purpose. Known volumes of DNA samples and a
series of standard DNA solutions (500,250,125,63,31 and 15 ng/6 JlI) are loaded
on the same gel. After migration the gel is photographed using short-wavelength
ultraviolet radiation. The intensity of fluorescence of the unknown DNA is compared with that of the DNA standards and the quantity of DNA in the sample is
estimated. A critical parameter is the "visual reading" of fluorescence.
We developed a simple technique to quantify DNA using the LightCycler System. Retinoic X Receptor B is a human single-copy gene mapping to chromosome
* Chantal Roudier (~) (email: chantal.roudier@medecine.univ-mrs.fr)
Laboratoire Immunorhumatologie, INSERM EMI 9940, Faculte de Medecine,
27 bd Jean Moulin, 13005 Marseille, France
** LightCycier is a trademark of a member of the Roche Group