To perform restriction digestion of DNA with EcoR I and BamHI enzymes.
Restriction enzymes are Nucleases which can cleave the sugar-phosphate backbone of DNA, found in bacteria. As they cut within the molecule, they are commonly called restriction endonucleases. They specifically cleave the nucleic acids at specific nucleotide sequence called Restriction sites to generate a set of smaller fragments .
Restriction enzymes form part of the restriction-modification system of bacterial cells that provides protection against invasion of the cell by foreign DNA – especially bacteriophage DNA. But the cells own DNA is not cleaved by these Restriction enzymes. This self protection is achieved by the help of the specific DNA methyltransferase enzyme which will methylates the specific DNA sequence for its respective restriction enzymes by transferring methyl groups to adenine or cytosine residues to produce N6-methyladenine or 5-methylcytosine. An interesting feature of restriction endonuclease is that they commonly recognize recognition sequences that are mostly palindromes - they shows the same forward (5' to 3' on the top strand) and backward (5' to 3' on the bottom strand) sequences. In other words, they are nucleotide sequences or complimentary strands that read the same in opposite direction.
Types of restriction and modiﬁcation (R-M) system:
Type I enzymes :- Type I restriction enzymes exhibit both restriction and DNA modification activities.They require the cofactors such as Mg2+ ions, S-adenosylmethionine (SAM) and ATP for their activity. The recognition sequences are quite long with no recognizable features such as symmetry. Type I restriction endo nucleases cleaves DNA at nonspecific sites and that can be 1000 base pair or more from recognition sequence. However, because the methylation reaction is performed by the same enzyme which mediates cleavage, the target DNA may be modiﬁed before it is cut. Because of these features, the type I systems are of little value for gene manipulation.
Type II enzymes :- Type II enzymes and their corresponding modification methyltransferases act as separate proteins. They have a number of advantages over type I and III systems. First, restriction and modiﬁcation are mediated by separate enzymes so it is possible to cleave DNA in the absence of modiﬁcation. Secondly, the restriction activities do not require cofactors such as ATP or S-adenosylmethionine, making them easier to use. They require only Mg2+ ions as cofactors. These enzymes are site-specific as they hydrolyze specific phosphodiester bonds in both DNA strands. Class II restriction endonucleases are generally used as the key material in molecular biology and recombinant DNA techniques, including genome mapping, RFLP analysis, DNA sequencing, and cloning.
Type III enzymes :- Like Class I enzymes, Type III enzymes possess both restriction and modification activities.They recognize specific sequences and cleave 25 - 27 base pairs outside of the recognition sequence, in a 3´ direction. They require Mg2+ ions for their activity.
Type IIs enzymes, have similar cofactors and macromolecular structure to those of type II systems, the fact that restriction occurs at a distance from the recognition site limits their usefulness.
The first three letters of the restriction enzyme refer to the organism from which the restriction enzyme was originally isolated, the fourth letter (if present) refers to the strain, and the Roman numerals serve as indices if the same organism contains several different restriction enzymes.
e.g. EcoR I and EcoR V are both from Escherichia coli, strain R; I and V are the order in which they were discovered.
BamHI Bacillus amyloliquefaciens, strain H, Ist enzyme
SmaI Serratia marcescens, Ist enzyme
HaeIII Haemophilus aegyptius, 3rd enzyme
Restriction Enzyme cleavage:
Class II restriction enzymes generate three types of DNA ends, all possessing 5´-phos-phate and 3´-hydroxyl groups:
a) Cohesive 5´ ends:- For example, ends generated by EcoR I:
b) Cohesive 3´ ends:- For example, ends generated by Pst I:
c) Blunt ends:- For example, ends generated by Hae III
Sticky ends (Blunt ends) are produced by cutting the DNA in a staggered manner within the recognition site producing single stranded DNA ends. These ends have identical nucleotide sequence and are sticky because they can bind to complementary tails of other DNA fragments cut by the same Restriction enzyme.
Isoschizomers and neoschizomers:
Different restriction enzymes, isolated from different organisms can have identical recognition sequences, such enzymes are called isoschizomers. Neoshizomers are Isoschizomeric enzymes but it cleaves at different recognition site.
Restriction enzymes are powerful tools of molecular genetics used to:
• Map DNA molecules
• Analyze population polymorphisms
• Rearrange DNA molecules
• Prepare molecular probes
• Create mutants
Factors affecting Restriction Enzyme Activity:
Temperature: Most digestions are carried out at 37°C. However, there are a few exceptions e.g., digestion with Sma I is carried out at lower temperatures (~25°C), while with Taq I at higher temperature i.e., 65°C.
Buffer Systems: Tris-HCl is the most commonly used buffering agent in incubation mixtures, which is temperature dependent. Most restriction enzymes are active in the pH range 7.0-8.0.
Ionic Conditions: Mg2+ is an absolute requirement for all restriction endonucleases, but the requirement of other ions (Na+/K+) varies with different enzymes.
Methylation of DNA: Methylation of specific adenine or cytidine residues within the recognition sequence of the restriction enzyme affects the digestion of DNA.
It is an alteration of the specificity of restriction enzyme mediated cleavage of DNA that can occur under some non standard conditions that differ from the optimum for the enzyme. This alteration leads to the cleavage at non specific sites.
Nonstandard conditions include:
1. High pH (>8.0).
2. Glycerol concentrations >5% (important, because enzymes are usually delivered as concentrated stock in 50% glycerol).
3. High concentration of enzyme (>100 U/µg of DNA).
4. Prolonged incubation time with enzyme.
5. Presence of organic solvents in the reaction (e.g., phenol, chloroform, ethanol,DMSO).
6. Incorrect cofactor (i.e., Mn2+,Hg2+or Co2+instead of Mg2+).
To avoid star activity, always use the optimal buffer system and enzyme amount recommended. Make sure that the DNA preparation is free of organic solvents and contaminating salts.
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