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In this article we will discuss about agarose gel electrophoresis.
To know about quality and quantity of genomic DNA, agarose gel electrophoresis is performed on genomic and amplified DNA. Larger molecules migrate more slowly because of greater friction drag and they form their way through the pores of gel, less efficiently than smaller molecules.
Range of separation in gel containing different amount of agarose is as indicated below:
The steps are as follows:
1. The open ends of the clean, dry plastic tray, supplied with the electrophoresis apparatus are sealed to form a mould. The mould is set on a plain horizontal table.
2. 0.8% agarose solution is prepared for genomic DNA electrophoresis and 1.8% for that of amplified DNA. 0.5 µg/ml ethidium bromide of 1 X TAE is added into the agarose after boiling. The boiling can be done either in microwave oven (about 10 min.) or water bath for about 30 min. till the solution becomes transparent. A comb with adequate number of wells (13 or 24 etc.), is inserted vertically into the gel before polymerisation to make the wells. The thickness of gel is about 1 mm.
3. After the gel is completely set, the comb is removed carefully and the gel is mounted over the electrophoretic tank containing 1 X TAE buffer in amount sufficient to immerse the gel in the buffer.
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4. The λ Hind III is used as marker DNA.
5. The DNA loading dye is mixed with DNA samples in ratio of 1: 5.
6. The sample is loaded in wells of gel with micropipette.
7. Electrophoresis is done at 100 volts till the dye front reaches the end of the gel.
8. The gel is documented through gel documentation system (e.g. Syngene-Gene Geneus etc.).
Advantages:
a. Non-radioactive detection
b. No prior DNA sequence information for genome required
c. Universal primers work in any genome
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d. Very small amounts of genomic DNA are sufficient
e. Multiplex detection of polymorphism
f. Experimental simplicity
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g. No need for expensive equipment beyond a thermal cycler and a trans-illuminator.
Limitations:
1. RAPD polymorphisms are inherited as dominant-recessive characters. This causes a loss of information relative to markers which show co-dominance.
2. RAPD primers are relatively short, a mismatch of even a single nucleotide can often prevent the primer from annealing, hence, there is a loss of band.
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3. RAPD is sensitive to changes in PCR conditions resulting in changes to some of the amplified fragments.
Applications:
a. Generating genetic maps
b. Mapping traits in segregating populations
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c. Mapping traits in near-isogenic lines
d. Mapping traits using bulk segregant analysis
e. Saturating regions of a genome with markers
f. Fingerprinting individuals
g. Germplasm analysis
h. Measurement of genetic distance between individuals
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i. Estimating relative parental contributions in backcross.
