Quantification of DNA

DNA in solution. The most simple and accurate is the

spectrophotometric method, but it has a relatively low sensitivity. If the total content of nucleic acids is small, the concentration of DNA can be determined from the intensity of their fluorescence in UV light after staining with ethidium bromide
a. Electrophoretic Method For an approximate evaluation of the concentration of DNA, different amounts of marker nucleic acids are applied to the gel. The concentration of DNA in the sample is evaluated by comparing the fluorescence intensity of the sample and standard markers with known concentrations. It is important that the DNA samples are compared with the corresponding markers, since for the same amount of DNA the fluorescence intensity in UV light differs.
B. Spot Method The concentration of nucleic acids in a solution can be determined by staining the solution with ethidium bromide by measuring the fluorescence intensity in UV light and comparing it with the fluorescence of markers of known concentration. As with the electrophoretic method, the markers must be nucleic acids of the corresponding type (i.e., DNA)

U)
are made up of alternating single
and double bonds.

Such ring structures absorb in
the U.V.
Each of the four nucleotide
bases has a slightly different
absorption spectrum, and the
spectrum of DNA is the average
of them.

a sample absorbs.



Consists of two instruments:
A spectrometer for producing

light of any selected color (wavelength)
A photometer for measuring the intensity of light

specific wavelength) passes through a solution there is usually a

quantitative relationship (Beer's law) between the solute concentration and the intensity of the transmitted light, that is, the more concentrated the specimen is, the less light is transmitted through it.

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the
amount of light absorbed.

Nucleic acid

(DNA/RNA)
Qualitative analysis Quantitative analysis
Size-Quality Purity- Yield
Agarose gel electrophoresis Spectrophotometry

the solution.
Determine if samples are useful for downstream applications like:

PCR Restriction digests.







Advantages of the nanodrop
It is a simple machine and economical on space.
Easy-to-use spectrophotometer
Can measure small volumes of DNA, RNA and
protein concentrations.

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sample purity (260:280).

Absorbance at 260 nm
Nucleic acids absorb UV light

at 260 nm due to the aromatic base moieties
within their structure. Purines (thymine, cytosine and uracil) and pyrimidines (adenine and guanine) both have peak absorbances at 260 nm, thus making it
the standard for quantitating nucleic acid samples.

Absorbance at 280 nm
The 280 nm absorbance is measured where proteins and phenolic compounds have a strong absorbance. Similarly, the aromaticity of phenol groups of organic compounds absorbs strongly near 280 nm.

Absorbance at 230 nm
Many organic compounds have strong absorbances at around 225 nm. In addition to phenol, TRIzol, and chaotropic salts, the peptide bonds in proteins absorb light between 200 and 230 nm.

analysed.







A260/230 ratio indicates the presence of organic contaminants, such as

(but not limited to): phenol, TRIzol, chaotropic salts and other aromatic compounds.

Samples with 260/230 ratios below 1.8 are considered to have a significant amount of these contaminants

Various nanodrop models are available e.g Nanodrop 8000, Nanodrop lite and Nanodrop 2000/2000c which is currently available at BecA labs.

to assess the purity of the samples (esp. RNA) by

analyzing the A260/280 and A260/230 ratios.

Quantification cannot be assessed by the NanoDrop because they are outside the lowest concentrations the NanoDrop is designed to measure.

A more sensitive method such as an Agilent Biolanalyzer or Qubit analysis is recommended.

proteins and nucleic acids, migrate in response to an electrical

field.
Their rate of migration through the electrical field, depends on the strength of the field, on the net charge, size, and shape of the molecules, and also on the ionic strength, viscosity, and temperature of the medium in which the molecules are moving.
As an analytical tool, electrophoresis is simple, rapid and highly sensitive.
It can be used analytically to study the properties of a single charged species or mixtures of molecules. It can also be used preparatively as a separating technique

rDNA procedures. A convenient method for both separation and purification

is gel electrophoresis. DNA molecules have a uniform charge to mass ratio. In an electric field they run towards the anode and are separated based on size (length) when electrophoresed through a gel sieving network made of polyacrylamide or agarose (Fig. 5.19a). DNA bands can be visualized by radiolabeling the DNA or by noncovalent binding of the fluorescent dye known as ethidium bromide (Fig. 5.19b). The region of the gel containing the band can be excised and the DNA fragment obtained by extraction with a buffer.

Separation of DNA Fragments by Gel Electrophoresis

buffer chambers containing separate electrodes so that the only electrical

connection between the two chambers is through the gel.

chemical structures, they both make porous gels.
A porous gel

acts as a sieve by retarding or, in some cases, by completely obstructing the movement of macromolecules while allowing smaller molecules to migrate freely.
By preparing a gel with a restrictive pore size, the operator can take advantage of molecular size differences among proteins

light at specific wavelengths.
Examples are fluorescein and rhodamine derivatives.

For sequencing applications, these molecules can be covalently attached to nucleotides.

labeling the sequencing ladder at the 5′ ends of the

chains.
In dye terminator sequencing, the fluorescent dye molecules are covalently attached to the dideoxynucleotides, labeling the sequencing ladder at the 3′ ends of the chains.

the four ddNTP.
Since the terminating nucleotides can be distinguished by

color, all four reactions can be performed in a single tube.

A

T

G

T

The fragments are distinguished by size and “color.”

T/A A/A

Automated Sequencing

Dye primer or dye terminator sequencing on capillary instruments.
Sequence analysis software provides analyzed sequence in text and electropherogram form.
Peak patterns reflect mutations or sequence changes.