HindIII is commonly isolated from the Haemophilus bacteria d Bacteria. It has a polymorphic restriction site on the intron 19 of factor VIII gene. It recognizes rthe double stranded sequence of the DNA at AAGCTT and then cleaves after A-1 (Dubey, Hussain & Mittal n.d.).DNA is negatively charged. The working principle in gel electrophoresis involves the movement of the DNA sample in the agarose gel. The difference in the base pairs impact a difference in the molecular weight of the DNA and so when the current is introduced at the cathodic end of the electrophoretic chamber the DNA will move to the anode with the difference in weight causing the bands to be formed at different locations Francois 2010). Our reference sample (λDNA) has a restriction site for the enzyme and so presence of matching bands with our sample DNA will show the presence of the wild type gene. In the case of no match, then we will rule out the presence of the wild type gene to the presence of the mutant gene.
The three samples will be run concurrently. The samples will be placed on the wells and when the power is switched on the movement will be monitored and images taken to do the comparison. With one sample being undigested and the other one digested we want to differentiate between the wild type and the mutant genes. With the wild type after digestion by the restriction enzyme HindIII the DNA is broken down and forms strands with 3236 base pairs and 1125 base pairs (Isaac & Stacey 1994).
This implies that due to the digestion process the bulk DNA has been broken down into lighter fragments and so with reduced molecular weight it will move faster across the electrophoretic gel. The restriction process takes place at an optimum temperature of 370C.
Dubey, A. O., Hussain, N., & Mittal, N., n.d., ‘HindIII-based restriction fragment length polymorphism in hemophilic and non-hemophilic patients,’ Journal of Natural Science, Biology and Medicine, 1 (1), pp. 25-28, doi: