Restriction Digest Protocol, Principle, Result

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Table of Contents

What is restriction digestion?

In molecular biology restriction digestion is used to prepare DNA for analysis or other processing. It is also known as DNA fragmentation. According to Hartl and Jones Restriction Digestion is a;

This enzymatic technique can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence at a particular location have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. The cleavage method makes use of an important class of DNA-cleaving enzymes isolated primarily from bacteria. These enzymes are called restriction endonucleases or restriction enzymes, and they are able to cleave DNA molecules at the positions at which particular short sequences of bases are present.

In 1970, Hamilton-Smith published a paper on the discovery and purification of the first restriction enzyme, or endonuclease, HindII; for which he was awarded the Noble Prize in medicine.
These enzymes are invaluable in molecular biology, cloning, genetic engineering, and multiple other scientific disciplines
The restriction enzymes cleave the DNA molecule at a specific site or specific nucleotide sequence, is known as recognition sequences or Restriction sites.
What are possible applications for restriction digestion? well, restriction digest is used in different analytical techniques such as, Restriction fragment length polymorphism (RFLP), Short tandem repeat polymorphism (AFLP), Amplified fragment length polymorphism (STRP).

Overview

To prevent the attack of bacteriophage, bacteria develop different enzymes which are known as endonucleases that chop up any foreign DNA.
Hence this enzyme restricts the bacteriophage infection, they are also termed “restriction endonucleases”.
In molecular biology restriction endonucleases are also known as molecular scissors due to their ability to cut the DNA into fragments.
These enzymes are mainly found in the cytoplasm of bacterial cell, hence the bacteria protects their DNA by methylating the adenine or cytosine bases. These methyl groups prevent the binding of restriction enzymes, but not the normal reading and replication of genetic information. While the bacteriophage DNA lacks these protective methyl groups and will be destroyed.
The restriction enzyme with the modification methyltransferase together develops a restriction modification (RM) system. There are mainly four types of RM systems which are distinguished based on subunit composition, kinds of sequences recognized and cofactors needed for the activity.
Restriction enzymes derive their name from the genus, species and strains of bacteria from where they are originated, followed by a number that refers to discovery orders. For example; EcoR I and EcoR V are both from Escherichia coli, strain R; I and V are the order in which they were discovered.

How type II restriction enzymes works

The Type II restriction enzymes recognize specific DNA sequences and cleave the DNA at fixed locations at or near the recognition sites. They act as dimers, each subunit recognizing the same 5 to 3′ nucleotide sequence in complementary DNA strands and hence are said to recognize palindromic sequences.
Before the restriction enzyme cuts at its specific recognition site on a long DNA, it binds to a site amidst a very large number of non-cognate sites. This protein is then translocated from the initial site to a specific site, which occurs by “jumping or sliding”.
At this recognition site in presence of Mg2+, enzyme undergoes a conformational change, which kinks the helix and cleaves the DNA producing ‘blunt’ or ‘sticky’ ends.
For e.g. Hae III produced by Haemophilus aegypticus cleaves straight across the double helix when it encounters the following sequence to produce blunt ends.

However, many restriction enzymes cut in an offset fashion to give sticky ends, which have protruding 5′ or 3′ ends with unpaired bases depending upon the restriction enzyme, e.g. EcoR I recognizes the following sequence and cleaves each backbone between G and A base residue giving 5′ protruding ends.

Pst I recognizes the following sequence, cleaves between A and G residue to give 3′ protruding ends.

The resulting sticky ends can base pair with any DNA molecule that has the complementary sticky ends to give a recombinant DNA molecule.
When multiple enzymes recognize a single recognition sequence is known as isochizomers. For example Sma I and Xma I. These can cleave the DNA at same or different position within the recognition sequence.
The frequency of cleavage varies on the possibility of the existence of recognition sequences. Therefore, an enzyme with longer recognition sequences cut less frequently and consequently produces large fragments than do enzymes with shorter recognition sequences.

Dot Blot Protocol, Principle, Definition.

Dot Blot is a simplified technique of western blotting, which is mainly used for the detection of proteins.

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Dot Blot Protocol, Principle, Definition.

Factors affecting Restriction Enzyme Activity

There are different factors that are influencing the enzyme activity such as;

Temperature: Most of the digest is performed at 37°C. 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 buffer is used as a buffering agent in incubation mixtures, which is temperature-dependent. Most restriction enzymes show activity at the pH range 7.0-8.0.
Ionic Conditions: For the activity of restriction endonucleases Mg2+ ion is required. The requirement of ions (Na+/K+) varies based on enzymes.
Methylation of DNA: Methylation of specific adenine or cytidine residues within the recognition sequence of the restriction enzyme affects the digestion of DNA.

Aim

To perform restriction digestion of λ DNA with EcoR I and Hind III enzymes.
To analyze the restriction pattern by agarose gel electrophoresis.

Restriction Digestion involves fragmenting DNA molecules into smaller pieces with special enzymes called Restriction Endonucleases commonly known as Restriction Enzymes (RE). Because of this property restriction enzymes are also known as molecular scissors.

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The restriction enzymes are named from the cellular strain from which they are isolated. Restriction enzymes recognize specific sequences in the double-stranded DNA molecule (for example GATATC) and then cut the DNA to produce fragments, called restriction fragments. The target site or sequence which the restriction enzyme recognizes is generally from 4 to 6 base pairs, arranged in a palindromic sequence.

Once it is located, the enzyme will attach to the DNA molecule and cut each strand of the double helix. The restriction enzyme will continue to do this along the full length of the DNA molecule which will then break into fragments. The size of these fragments is measured in base pairs or kilobase pairs (1000 bases).

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Material Required for Restriction Digest

2X Assay buffer
λ/Mlu I digest
2.5X Gel loading buffer
Lambda DNA (substrate)
Restriction Enzymes: EcoR I, Hind III
Control λ DNA
Agarose
6X Staining dye
50X TAE
1.5 ml vials
Equipment: Dry bath, Gel rocker (optional).
Glassware: Beakers, Conical flask, Measuring cylinder, Staining tray.
Distilled water.
Other: Crushed ice/Genei Cooler, Tips, Micropipette.