Question

State several reasons why we must quantitate the amount of DNA that we isolate. Then state and describe 5 ways that we can quantitate DNA and state which ones are specific for human DNA.

Answer 1

Quantification of DNA is a very important step in many procedures where it is necessary to know the AMOUNT of DNA that is present when carrying out restriction digests or performing different techniques such as PCR and RAPDs. Quantification also helps to know the quality and purity of the DNA.

There are several methods for quantifying DNAs

UV Spectrophotometry;

Absorbance has been the method of choice for routine quantification of DNA and RNA since decades. It is simple and convenient to use as no further sample treatment (other than DNA extraction) or reaction with other substances is required. However, it is not very specific (it measures all nucleic acids as a whole) and sensitive to contaminants, so it demands very pure DNA to be accurate.

The diphenylamine method :

Another method of quantifying DNA by absorbance is the diphenylamine method, which is based on the reaction of diphenylamine with deoxyribose sugar to form a blue complex. This method is time-consuming, has a low sensitivity and is therefore no longer used in most laboratories

Based on flourescence ;

The use of fluorescent dyes permits the quantification of DNA with much higher sensitivity than measuring absorbance of DNA itself (typically 10-100 times higher, depending on the specific methods compared). In addition, specific dyes can be used to stain only specific types of nucleic acid, such as dsDNA or RNA, thereby increasing the specificity of the quantification and reducing the effect of contaminants. However, fluorescence-based methods are more expensive than measuring absorbance at 260 nm and often require a standard curve to be prepared.

Electrophoresis :

If the DNA to be quantified is a plasmid, gel electrophoresis is a popular method. In this method, a fluorescent dye such as ethidium bromide or SYBR Green is added to the gel or the samples, followed by electrophoresis of the samples parallel to a DNA ladder and the bands in the gel are visualized with a transilluminator or a gel documentation system. The bands of the DNA ladder have a known mass, so that the amount of DNA in our samples can be estimated by comparing the intensity of their bands with that of the mass ladder. While the method is very specific and ignores most impurities (since the DNA of interest has been separated from other nucleic acids or contaminants by electrophoresis), it is time-consuming, inaccurate (since quantification is performed visually or using image analysis software) and has low sensitivity.

Based on PCR :

It is also possible to use quantitative real-time PCR (qPCR) and digital PCR or Droplet Digital PCR (ddPCR) to quantify DNA. Digital PCR even enables nucleic acid quantification without a standard curve, since the molecules are separated according to the Poisson distribution. This results in a digital event in each reaction vessel (chip or droplet), so that the proportion of reaction vessels in which amplification has taken place is directly proportional to the amount of DNA sequence used, and thus the amount can be determined directly. The procedure is very sensitive, but requires a special and expensive apparatus as well as a relatively complex experimental setup and more time than the other methods described.

Fluorescent based methods are specific but are expensive.