DNA Fingerprinting Simulation Using RFLP Gel Electrophoresis

Theoretical Background/Context

• 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. T
• 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.
• This DNA fingerprinting interactive process enables students to understand the relationship between restriction enzyme recognition sites and the resulting fragment patterns.

Principle Work of DNA Fingerprint Simulation

• Agarose is isolated from the seaweed genera Gelidium and Gracilaira.
• 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 DNA fingerprinting procedure, 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..)

4) Voltage 

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.
• This visualization step is crucial in DNA fingerprinting using gel electrophoresis to reveal the banding patterns.
• 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 DNA fingerprinting lab 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,  and understanding their role in electrophoresis in DNA fingerprinting is essential for accurate DNA analysis. 
• The combination of gel electrophoresis and dna fingerprinting techniques in this dna fingerprint simulation provides a comprehensive understanding of molecular identification methods.

EcoRI:

5’ G^AATTC 3’

3’ CTTAA^G 5’

HindIII:

5' A ↓AGCTT 3' 

3' TTCGA ↑A 5