Restriction Enzymes: Overview & Application

Restriction Enzymes: Overview & Application

Restriction Enzymes: Overview

Restriction enzymes are a type of nuclease enzyme that recognizes a specific DNA sequence/recognition site and cleaves at or near that specific site. The site recognized by the enzyme is generally 4-8 base pairs long.

The enzymes were first discovered by a scientist named Werner Arber when he was studying host-controlled restriction of bacteriophages. He proposed that Escherichia coli or E. coli (and other bacterial cells) could defend themselves against foreign DNA using a genetic defense mechanism catalyzed by enzymes.

DNA molecules consist of two complementary nucleotide strands that form a helical structure. The restriction enzymes identify the recognition sequence and cleave the nucleotide strands at the restriction site that results in DNA fragments. However, the ends of the cut DNA are never the same. It differs based on the type of enzyme that cleaves the nucleic acid strand.

For example, SmaI cleaves double-stranded DNA into restriction fragments having blunt ends or flat ends. However, the enzyme EcoRI cuts the target DNA in a way that the ends of the resulting two restriction fragments have overhangs of the nucleotide sequence.

This type of end is known as the sticky end because they are complementary to each other and easily bond with their complementary DNA strands. Similarly, the HindIII enzyme also cleaves DNA in a way that results in sticky ends.

Figure: The representation of a restriction map of Bacteriophage lambda.

The ends are then sealed by the DNA ligase enzyme, which also glues together newly synthesized DNA during DNA replication processes to provide connected DNA strands.

In this article, we will cover the types of restriction endonuclease, how they work, their functions, and their applications in life sciences labs and industries.

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How Do Restriction Enzymes Work?

Restriction enzymes cut DNA strands by identifying a short specific sequence, known as the recognition site. As the identification is done, the enzyme wraps around the double-stranded DNA molecule and cleaves it by hydrolyzing the sugar-phosphate backbone between nucleotides of the two complementary strands.

Each restriction enzyme has its own unique recognition sequence of 4-8 nucleotides and most of them are palindromic sequences (reads the same from forward and backward).

Figure: An illustrative diagram of DNA cleavage by the restriction enzyme.

Bacterial cells protect their own DNA from getting degraded by the restriction enzyme using methylases enzyme that attaches methyl groups to the cytosine and adenine bases of their recognition site.  

Type of Restriction Enzymes

Based on their enzyme cofactor requirements, the nature of their target sequence, composition, and the DNA cleave site relative to the target sequence, the restriction enzymes are categorized into five groups:

  • Type I restriction enzymes: Cleaves at a site distant from the recognition site. To perform the cleavage activity it requires S-adenosyl-L-methionine and ATP. Further, it also requires multifunctional proteins having methylase and restriction digestion activities.
  • Type II restriction enzymes: Either cleaves within the sequence of the recognition site or close to it. To perform its cleavage activity it requires magnesium as a cofactor and single-function protein, independent of methylase enzyme.
  • Type III restriction enzymes: Cleaves at a distance close to the recognition site. It requires ATP and S-adenosyl-L-methionine as cofactors to carry out restriction digestion and methylation activities. It’s also known as modification methyltransferase.
  • Type IV restriction enzymes: Targets modified DNA, such as hydroxymethylated, methylated, and glucosyl-hydroxymethylated DNA. A good example of this is the McrBC and Mrr systems of E. coli or Escherichia coli.
  • Type V restriction enzymes: Utilizes guide RNA to target specific target sequences from the invading pathogens (such as Phage DNA in E.coli). CRISPR-Cas9 is one of the most popular enzymes in this category.  

What are Restriction Enzymes Used For?

Restriction enzymes are one of the most common and popular tools used in biotechnology and molecular biology labs in several workflows involving assays like gel electrophoresis, sequencing, southern blotting, cloning, and PCR reactions.

Building a recombinant plasmid

Restriction enzymes are widely used for cloning purposes. First, the correct restriction enzyme needs to be chosen so that the target gene and plasmid are both cut at the same sequence. This results in the target gene and plasmid having the same kind of sticky ends (For example, if cut with EcoR1, they will both have complementary sticky ends). Then, the DNA ligase enzyme joins the two fragments together by base pairing. And, this is how a recombinant plasmid is formed for cloning purposes.

Figure: An illustrative diagram of recombinant plasmid DNA creation.

What Industries Use Restriction Enzymes?

Restriction enzyme digestion is extensively exploited in labs for a range of workflows in combination with assays like sequencing, gel electrophoresis, or polymerase chain reactions.

Molecular Biology

Gene alleles can be easily distinguished using restriction enzymes by specifically recognizing changes in a single base of DNA (or single-nucleotide polymorphisms (SNPs). Furthermore, the enzyme also has applications in mutation, gene expression, and population polymorphism studies.

Criminal Forensics

Restriction endonuclease digestion is widely used for DNA fingerprinting in criminal forensics. Here, DNA is isolated from the samples obtained from the victim, the suspect, and the evidence obtained on the criminal site. Then, they are digested using restriction enzymes followed by DNA fragment separation through agarose gel electrophoresis.

The obtained bands are transferred on nylon membranes and treated with radioactively labeled probes, which provides a clear visualization of the matched DNA fragments.

Genetic Engineering

A restriction enzyme is one of the most essential tools in genetic engineering. Apart from inserting target genes in organisms and creating genetically modified organisms, they are also used as probes to identify the exact locations of some specific genes or DNA sequences in an organism’s genome.  

It is also possible to generate a DNA map by restriction digest that illustrates the relative positions of the genes. Furthermore, they also have an application in digesting genomic DNA for gene analysis by using the southern blotting technique.

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A restriction enzyme is a protein originally found in bacterial species that cleave DNA strands at a specific cleavage site. Several laboratory techniques, such as recombinant DNA technology and genetic engineering, require restriction enzymes. It has profound applications in cloning, DNA fingerprinting, and DNA mapping.

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