Gel Electrophoresis is a procedure used in molecular biology to separate and identify molecules (such as DNA and RNA) by size. The separation of these molecules is achieved by placing them in a gel made up of small pores and setting an electric field across the gel.
The molecules will move based on their inherent electric charge (i.e., negatively charged molecules move away from the negative pole) and smaller molecules will move faster than larger molecules; thus, a size separation is achieved within the pool of molecules running through the gel. The gel works in a similar manner to a sieve separating particles by size; the electrophoresis works to move the particles, using their inherent electric charge, through the sieve.
DNA fingerprinting allows the comparison of DNA from different organisms and the identification of a particular individual. Basically, DNA is extracted and cut into fragments using restriction enzymes. The fragments form a pattern on agarose gel electrophoresis according to their length. The longer the DNA fragments are generated, the larger their molecular weight and the shorter they travel in an electrophoresis setting. This pattern looks a lot like the barcode on products in the supermarket and it resembles the individual’s DNA fingerprint.
To interpret the pattern formed, one must realize that restriction enzymes cut the DNA at specific base-pair sequences called recognition sequences. There are hundreds of restriction enzymes, each having a specific recognition sequence made of four to twelve base pairs. The lengths of the fragments generated by a restriction enzyme digest of an extracted DNA sample depend upon the number of cuts made and their locations. This depends on the number of recognition sequences of the enzyme used on the extracted DNA and their locations. Thus, everyone’s DNA is cut by restriction enzymes into distinctive different sized fragments that appear on the electrophoresis gel in the form of bands.
Agarose is isolated from the seaweed genera Gelidium and Gracilaria. It’s mixed with a buffer and heated in a microwave, then left to cool down before pouring in the cast. A comb is added at a specific site to form the wells required for sample upload. Then the gel is left to solidify.
The concentration of gel = weight of agarose/volume of buffer (g/ml). For a standard agarose gel electrophoresis, 0.8% gel gives good separation of large 5–10kb DNA fragments, while 2% gel gives a good resolution for small 0.2-1 Kb DNA fragments.
During gelation, agarose polymers associate non-covalently and form a network whose pore sizes determine a gel's molecular sieving properties.
The phosphate in the backbone of DNA is negatively charged, therefore DNA fragments will migrate to the positively charged anode.
DNA has a uniform mass/charge ratio, therefore DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight.
The rate of migration of a DNA molecule through a gel is determined by the following:
1) size of DNA band (the heavier, the slower)
2) agarose gel concentration (usually 0.8%)
3) DNA conformation (linear/plasmid/etc..)
5) Electrophoresis buffer.
6) Ethidium bromide: EtBr is positively charged, thus; reducing the DNA migration rate by 15%. Other stains for DNA in agarose gels include SYBR Gold, SYBR green, Crystal Violet and Methyl Blue.
UV light activates electrons in the aromatic ring of ethidium bromide releasing light as electrons return to the ground state. EtBr intercalates itself in the DNA molecule in a concentration-dependent manner. So higher intensity means a higher amount of DNA.
Restriction enzymes are present in bacteria. They are named according to the bacteria from which they are extracted. They protect bacteria by digesting foreign DNA at specific sequences.
Restriction enzymes such as (EcoRI) and (HindIII) are used in this experiment to Cut the extracted DNA. EcoRI recognizes the 6 bp sequence 5’ GAATTC 3’ and makes a staggered cut between the G and A creating sticky ends. HindIII recognizes the 6 bp sequence 5’ AAGCTT 3’ and makes a staggered cut between the A and A creating sticky ends. Both enzymes cut best at 37°C.
5’ G^AATTC 3’
3’ CTTAA^G 5’
5' A ↓AGCTT 3'
3' TTCGA ↑A 5'