Restriction analysis



Agarosegel electrophoresis is a way of separate DNA fragments in a given DNAstrand. The techniques use generally can be studied using pUC19DNAand plasmids, DNA plasmid mapping, restriction enzyme and UVtransillumination.


Setup of digestion


Tableand band distances

Discussionand analysis

Restrictionenzymes are naturally occurring bacterial endonucleases thatrecognize a large range of DNA sequences and cleave to the sequenceresulting in modifications of the sequence[ CITATION add15 l 1033 ]. They are able to cut through the nucleotides of such a sequencethereby allowing for a recombination of the DNA and giving a newsequence to the strand. Contemporary DNA technologies are using suchtechniques in different fields in our daily lives to develop newstrains of organisms or make decisions. New varieties of an organismcan be developed resulting in acquisition of new qualities that maynot have existed earlier on. Cloning is a common practice today andhas resulted in improved scientific technology in specific fields ofscience.

Inlooking at enzymatic modifications of DNA, a negative controlexperiment is conducted purposely to give a mapping that shows theDNA concentrations and locations on a given DNA strand. Tube 1 inthis experiment is the negative control set up since it does not havethe restriction enzymes. Lane 2 in the map gives the strandsobtained from the negative control which is used for comparison withthe bands in the other lanes other than lane 1. Lane 1 gives theladder for the set up depicting the uncut DNA bands. The multiplebands in the uncut DNA lane indicate migration of the tracking dyesafter being analyzed on the agarose gel[ CITATION Cab15 l 1033 ].

Itis expected that those tubes that are having the enzymes will give amap showing bands in different positions and different distances ascompared to the negative control. The enzymes will digest and adjustthe DNA sequence to give a new sequence. In this experiment, themapping clearly shows bands in new positions and different distancesdepending on the enzyme or a combination of the enzymes used. Thisindicates that enzyme digestion has occurred.

However,incomplete digestion and experimental errors can be noticed bycomparing the distribution of the strands after digestion with thedistribution of the strands in the control lane. In this experiment,there could be some errors involved as seen in lane 3, lane 5 andlane 6. A combination of the restriction enzymes Hind II and Bg1gives new bands that are not evident in digestions under the enzymeswhen applied singly in the experiment. It is possible that this is aresult of an experimental error or incomplete digestion. Maps forthe digestion reaction of enzyme PvU11 and a combination of thisenzyme with Hind II and Bg1 are consistent bands as seen in lane 7and lane 8.

Restrictionenzymes are many and since they cleave to specific sites in a DNAsequence, restriction digests have become the most widely used methodscientists employ to selectively move a specific piece of DNA fromone plasmid to another[ CITATION add15 l 1033 ].In addition to this, this journal states that a diagnosticrestriction enzyme digest takes advantage of the fact thatrestriction enzymes cleave DNA at specific sequences calledrestrictions sites. This restriction sites are the targets ofmodifications in the DNA of an organism.

Thisexperiment on restriction enzymes is an avenue for development of newgenes in an organism. With this in mind, we can improve plant andanimal varieties for better agricultural yields free of genes thatare poor in quality. On the other hand, we can also change asequence to result in a self destructive gene especially in caseswhere an organism is detrimental to human life. However, legalissues must also be looked into before these scientific technologiescan be profoundly used in our daily lives. Note that restriction inthe cellular economy has advanced by leaps and bounds in recent years[ CITATION TAB15 l 1033 ].


addgene.org. &quotProtocol-How to perform a diagnostic digest.&quot Addgene website. https://www.addgene.org/plasmid-protocols/diagnostic-digest/ (accessed April 27, 2015).

Bickle, T.A. &quotbiology of DNA restriction.&quot asm.org. http://mmbr.asm.org/content/57/2/434.short (accessed april 27, 2015).

Cabrillo.edu. &quotDNA,Restriction enzymes and gel electrophoresis.&quot Cabrillo web site. https://www.cabrillo.edu/~ytan/Bio1AF04/Lab8%20DNA%20and%20Rest%20lab.pdf (accessed April 27, 2015).