Restriction Enzymes Overview

Study Notes

Restriction enzymes are molecular scissors that cut double-stranded DNA molecules at specific points.
They are found naturally in prokaryotes.
They are important tools for manipulating DNA.
These enzymes were discovered in bacteria
They help bacteria destroy viral DNA.
Over 3,000 enzymes have been identified.
Around 200 have unique properties.
Many are commercially available.
Researchers rely on restriction enzymes for many laboratory processes.
Key applications in research, medicine, and agriculture; DNA profile analysis, disease diagnosis. paternity/family relationship testing and forensics.

Enzymes are biological catalysts found in all cells.
Enzymes have unique chemical structures; they only act on specific substrates.
Optimal enzyme function requires specific conditions (temperature, pH).
Enzymes are critical for various cellular processes, including digestion, DNA replication, protein synthesis.
Disruption to these conditions disrupts the unique chemical structure of enzymes.

A recognition sequence is a DNA sequence where a DNA-binding domain exhibits binding specificity.
Recognition sequences are palindromes.
A recognition site is the specific position of the recognition sequence within the DNA molecule.

DNA sites are made up of four to eight nucleotides.
Palindromic DNA have sequences that read the same forward and backward when read from the corresponding strands.
Specific restricted enzymes cut specific DNA sequences.
Example of a recognition sequence: GAATTC
Resulting pieces of DNA are called restriction fragments.

Type I: Random cleavage, 1000bp from recognition site. Endonuclease and methylase are located on a single protein molecule.
Type II: Specific cleavage, within recognition site. Endonuclease and methylase are separate entities.
Type III: Random cleavage, 24-26 bp from recognition site. Endonuclease and methylase are located on a single protein molecule.

Arbor and Dussoix in 1962 discovered that certain bacteria contain enzymes called endonucleases with the ability to cleave DNA.
Smith and colleagues in 1970 purified and characterized the cleavage site of a restriction enzyme in Hemophilus influenzae.
Werner Arber, Hamilton Smith, and Daniel Nathans shared the 1978 Nobel Prize for Medicine and Physiology for their discovery of restriction enzymes.

Most bacteria use restriction enzymes as a defense mechanism against bacteriophages (viruses that infect bacteria).
Restriction enzymes prevent phage replication by cleaving phage DNA at specific sites.
The host's DNA is protected by methylases that add methyl groups to adenine or cytosine bases within the recognition site, thereby modifying the site and protecting the host DNA.

Sticky ends: Protruding ends of the fragments that are generated by restriction enzymes, which can easily rejoin with matching sticky ends.
Blunt ends: Generated when restriction enzymes cut the DNA at symmetrical sites.
These ends are suitable for joining fragments with matching blunt ends.

Prepare a Master Mix which contains the buffer and enzyme.
Add the Master Mix to the DNA sample, mix thoroughly.
Incubate at appropriate temperature and time, which depends on the enzyme being used.
View the result by gel electrophoresis.

Step 1: Extraction and purification of DNA from cells.
Step 2: Digestion with Restriction Enzymes to cut the DNA at specific sites.
Step 3: Gel Electrophoresis to separate the DNA fragments based on size.
Step 4: Southern Blot and hybridization with a radioactive probe to identify specific DNA fragments.
Step 5: Analysis of the fragment distribution to discern the pattern or map of the fragments.

Aim: to separate and identify the digested fragments.
Separation by agarose gel electrophoresis.
DNA is negative and migrates to cathode.
Process of restriction mapping involves sample DNA, single and double restriction digestion, agarose gel electrophoresis and analysis of bands.
Length of DNA to be digested affects the number of fragments being produced.

Smith and Nathans (1973) established a three-letter abbreviation system using the host organism's name
First letter of genus, followed by two letters of species.
Abbreviation in italics.
Different enzymes from the same host can be distinguished using roman numerals, e.g., EcoRI, EcoRII.

Isoschizomers: Restriction enzymes that recognize the same DNA sequence. The cut sites may or may not be identical.
Neoschizomers: Enzymes that recognize the same DNA sequence with different cut sites relative to the same sequence.

Frequency depends on base composition and length of recognition sequence.
DNA Molecules and Bonds: DNA molecules hold genetic info and are composed with phosphodiester bonds.

Nucleases are enzymes that break phosphodiester bonds in DNA. (hydrolyzing)
Classified into endonucleases (cuts in the middle) and exonucleases (cuts at the ends).
Restriction enzymes are endonucleases.