Vietnam: Methods for determining DNA concentration in solutions

Vietnam: Methods for determining DNA concentration in solutions (Internet image)

According to the Appendix on technical procedures for qualitative and quantitative analysis of Deoxyribonucleic Acid issued with Circular 13/2013/TT-BTNMT, the commonly used methods to determine DNA concentration in solution are stipulated:

Electrophoresis Method: DNA is separated using the electrophoresis method on an agarose gel matrix based on its charge and molecular weight. After electrophoresis, the gel matrix is stained with Ethidium Bromide (EtBr). EtBr binds to DNA molecules and emits orange fluorescence when excited by UV light. Since the fluorescence intensity is proportional to the total amount of DNA, the quantity of DNA in the sample can be estimated by comparing the fluorescence emitted by the unknown sample with a standard quantitative series.

Spectrophotometric Measurement of DNA Contents: Nucleic acids in solution absorb ultraviolet (UV) light in the range of 210 nm to 300 nm with a maximum absorption at 260 nm. Since DNA, RNA, and nucleotides all have maximum absorption at 260 nm, DNA cannot be specifically determined using a UV spectrophotometer if the solution is contaminated with nucleotides and RNA. Therefore, RNA must be removed by hydrolysis with RNase enzymes. The nucleotides and oligonucleotides resulting from RNA hydrolysis also need to be removed, otherwise, they could lead to an overestimation of the DNA content in the test sample. Additionally, double-stranded DNA absorbs less UV light than single-stranded DNA, so the absorption coefficient should be considered when calculating concentration.

Periodic calibration of the spectrophotometer should be conducted by measuring the concentration of control DNA solutions.

More details can be found in Circular 13/2013/TT-BTNMT, which comes into effect in Vietnam from August 5, 2013.

Le Vy

>> CLICK HERE TO READ THIS ARTICLE IN VIETNAMESE