Restriction digestion Restriction digestion employs the function of one or more restriction enzyme to selectively cut DNA strands

into shorter restriction fragments. There are many types of restriction digestion cuts that can be generated which are more suitable for analytical techniques such as chromatography. Restriction digests are performed in Genetic fingerprinting techniques using restriction fragment length polymorphisms, and are used in many other DNA manipulations.

Restriction digestion mechanism Each specific restriction enzyme cuts DNA segments within a specific nucleotide sequence, and almost always breaks the DNA at recognized DNA sequences (the restriction enzyme recognition sequence) to produce the same ends every time. These recognition sequences are usually 7 nucleotides long however they can range from four to twelve nucleotides long. As there are four nucleotides - A,C,G and T - exact DNA sequences of four or eight or twelve nucleotides are only found throughout the genome or a given DNA sequence. Hundreds of restriction enzymes have been isolated from bacteria that are able to digest hundreds of specific sequences. As a result of this, one can obtain restriction enzymes that can cut most DNA fragments in the desired way. On the other hand, if the desired restriction sites are not present, they can be inserted into the DNA sequence using PCR or other methods. DNA fragment of interest can be clone into a plasmid or vector which contains many restriction enzyme sites within the multiple cloning site or MCS. Once the cuts are made with the restriction enzymes, the plasmid can then be loaded into a gel for electrophoresis. In electrophoresis, the negatively charged DNA is pulled through an agarose gel, causing the shortest fragments of the plasmid to extend farther into the gel, and separating each cut segment from the other. A marker is also loaded with the gel, indicating the amount of base pairs in each segment of the plasmid. The new DNA fragment may then be extracted from the gel by cutting it, and doing gel purification. Through ligation of a vector which has also been cut by a restriction enzyme, the fragment of interest can then be inserted into this vector, and a new

synthetic plasmid is formed. The new plasmid is then transformed into a biological system, and replication occurs. ( http://www.molecularstation.com/wiki/Restriction_digestion)
(http://www.molecularstation.com/dna/restriction-enzyme-digestion/)

Restriction Enzymes Restriction enzymes, also known as restriction endonucleases, are enzymes that cut a DNA molecule at a particular place. They are essential tools for recombinant DNA technology. The enzyme "scans" a DNA molecule, looking for a particular sequence, usually of four to six nucleotides. Once it finds this recognition sequence, it stops and cuts the strands. This is known as enzyme digestion. On double stranded DNA the recognition sequence is on both strands, but runs in opposite directions. This allows the enzyme to cut both strands. Sometimes the cut is blunt; sometimes the cut is uneven with dangling nucleotides on one of the two strands. This uneven cut is known as sticky ends. (http://www.gbiosciences.com/EducationalProducts/DNARestriction-Digestion-Analysis.aspx)

Figure 1: Cutting Type

HindIII

HindIII is

type

II

site-specific

deoxyribonuclease restriction

enzyme isolated

from Haemophilus influenzae that cleaves the palindromic DNA sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis. The cleavage of this sequence between the AA's results in 5' overhangs on the DNA called sticky ends:

Table 1: HindIII characteristic Enzyme HindIII Source Haemophilus influenzae Recognition Sequence 5'AAGCTT 3'TTCGAA 5'---A Cut AGCTT---3' A---5'

3'---TTCGA

Restriction endonucleases are used as defense mechanisms in prokaryotic organisms in the restriction modification system. Their primary function is to protect the host genome against invasion by foreign DNA, primarily bacteriophage DNA. There is also evidence that suggests the restriction enzymes may act alongside modification enzymes as selfish elements, or may be involved in genetic recombination and transposition.

EcoRI EcoRI is an endonuclease enzyme isolated from strains of E. coli, and is part of the restriction modification system. In molecular biology, this restriction enzyme creates sticky ends with 5' end overhangs. The nucleic acid sequence where the enzyme cuts is GAATTC, which is a palindrome as the complementary sequence is CTTAAG. It was generally used in a wide variety of molecular genetics techniques including cloning, DNA screening and deleting sections of DNA in vitro. Restriction enzymes like EcoRI that generate sticky ends of DNA are often used to cut DNA prior to ligation, as the sticky ends make the ligation reaction more efficient.

Table 2: EcoRI characteristic Enzyme EcoRI Source Escherichia coli Recognition Sequence 5'GAATTC 3'CTTAAG 5'---G Cut AATTC---3' G---5'

3'---CTTAA

Reagent Reagents used for the experiment in order to prepare the reaction mixture was consist of distilled water, 10x Bovine serum albumin, 10x buffer of salt concentration, restriction enzyme (concentration of U/l) and desired DNA (100 ng/l). The reaction mixtures need to be preparing for each restriction enzyme that to be in this experiment which is HindIII and EcoRI. The amounts of each reagent are shown in Table 3 below:

Table 3: Reagent for Reaction Mixture Reagents Distilled Water 10x BSA 10x Buffer (Salt concentration) Restriction Enzyme (10 U/ l) DNA (100 ng/ l) Total Amount 13 l 2 l 2 l 1 l 2 l 20 l

Agarose Gel Electrophoresis

Agarose gel electrophoresis is a process that undertakes biochemistry and molecular biology understandings to identify and analyse DNA and RNA strands. This is done by separating the genetic material by its size.

The genetic material is placed in the solidified wells of the agarose (a linear polymer composed of alternating isomers of the sugar galactose) at the cathode end. The negatively charged nucleic acid molecules move through the agarose matrix with the assistance of an electric field (electrophoresis). This is because genetic material is negatively charged, and will move towards the anode when current is passed through. The shorter molecules migrate faster than the longer molecules.

The use of electrophoresis buffer in the making of the agarose gel is to establish a constant pH and to provide ions to support the conductivity. If instead water was used, then the genetic material will not migrate during the electrophoresis.

The amount of voltage used is crucial to the migration of the genetic material. When increasing voltage is applied to the gel, larger fragments migrate proportionally to that of the smaller fragments. Thus, the voltage applied is usually 5 volts per centimetre to the gel. The gel is then immersed in ethidium bromide, a fluorescent dye that covalently binds (intercalates) between the bases of nucleic acid. Then UV light is passed through the gel to make the genetic material visible.