The process of cleaving DNA at a particular location with the help of the specific type of restriction endonuclease enzyme which helps in mapping, polymorphism study and studying mutation called as restriction digestion.
For understanding the process of the restriction digestion we have to first understand several terminologies such as what is digestion, what is DNA digestion, what is restriction endonuclease and what are the nucleases.
The core value of the present article is on the process of the restriction digestion and its role in the detection of mutation. Also on how it is applicable in the disease and how we can analyse the results of the restriction digestion.
Furthermore, we will interpret and analyse the results of the restriction digestion.
The content of the article:
- What is DNA digestion?
- What is restriction digestion?
- What are nucleases?
- The in vitro process of restriction digestion
- The factors affecting restriction digestion
- Methods of restriction digestion
- Restriction digestion results analysis
- Application of restriction digestion
Restriction endonucleases cut or cleave DNA at a specific location at their specific recognition site. The first restriction endonuclease was discovered into the year 1970 (HindII) by Smith, Thomas Kelly and Kent Wilcox. However, the term was first explained by W. Arber in the year 1960.
Nathans, Smith and W. Arber were awarded Nobel prize in 1978 for discovering the endonuclease.
“Endonucleases are DNA manipulating tool, a molecular scissor found in wide varieties of prokaryotes, especially, in bacteria.”
Interestingly, the phenomenon of endonuclease in bacteria was first explained in the 1950s by Salvador Luria and Giuseppe Bertani.
Around the era of the 1980s, the idea of cutting DNA with the help of the specific type of endonuclease was more clear and due to this the era of the genetic engineering was evolve. different techniques of genetic engineering such as gene editing, CRISPR-CAS9, genetic manipulation, DNA sequencing and DNA microarray was simultaneously evolved as we.
What is DNA digestion?
The process of cutting or cleaving DNA into smaller fragments with the help of the chemicals, physical and enzymatic methods are called the process of the DNA digestion.
The process of the DNA digestion creates mutation into the Genome. UV radiation, gamma rays, Heat and centrifugation are several physical methods which cut DNA vertically as well as horizontally.
Whereas, Hydroxyurea, camptothecin are chemicals that cut DNA horizontally.
Nucleases such as endonucleases and exonuclease are the enzyme used to cut the DNA, however, it cuts DNA vertically.
In the vertical cutting, the phosphodiester bonds between the adjacent nucleotides are broken whereas in horizontal cutting the hydrogen bonds between the nucleotides of the opposite strands are broken.
“The helicase cleaves the DNA horizontally by breaking the hydrogen bond between two strands during the replication while the endonuclease cut DNA vertically by breaking the phosphodiester bond.”
The polymerase chain reaction is the best example of the method for horizontal cutting while restricting digestion is the best example of vertical cutting.
Nonetheless, the process of DNA digestion is actually the process of vertical cutting, contrary to the denaturation process of the PCR.
Protein digestion is another method in which the complex molecules of the proteins are digested with the help of the special type of the enzymes such as trypsin, chymotrypsin and pepsins.
These enzymes cut the bonds between the amino acids and release the complexity of the protein molecules.
What are the nucleases?
Nucleases are the special types of enzymes (made of the proteins) which has the ability to cleave DNA at a specific location. The location at which the nuclease recognise the DNA is named as recognition site whereas the cutting site is called a restriction site.
The recognition site is the sequence on the DNA which is identified by the nuclease for the nuclease activity. Two types of nucleases are most common viz, exonuclease and endonuclease.
The exonucleases are the enzymes that cut the nucleic acid outside. It cuts the nucleotides at either 3’ or at 5’ ends of the DNA or on both ends of the DNA. The exonuclease can not cut DNA at a specific location into the whole genome.
On the other side, the endonuclease is the types of a nuclease which cut DNA specifically at any position into the whole genome (at where its recognition sites are located).
“Nuclease enzyme is one of the tools in the gene editing technique such as CRISPR-CAS9.” Read more on gene editing and CRISPR-CAS9 here,Gene editing and CRISPR-CAS9
What is restriction digestion?
The restriction endonucleases are involved in the bacterial and prokaryotic defence mechanism and hence the restriction digestion too. The virus and phages always target bacterial DNA.
The phage attacks the bacterial cell and destroys it. The endonucleases recognise the foreign DNA, bind on it and attack it, by doing this it cut the foreign DNA into small pieces and destroys vital genes of the phages.
The endonuclease attacks the foreign DNA and cleaves the phosphodiester bond between the adjacent nucleotide on both the strands. See the figure,
Numerous REase attacks numbers of different sequences present into the foreign DNA and protect the bacterial DNA from the attacks of the phages and other viruses.
Interestingly, a question arises in your mind that the DNA is the same in all organisms than how the bacteria protect their own DNA from the REase attacks or can REase of bacteria kills their own DNA?
The answer is yes it can kill their own DNA but thanks to the methylation.
Here the process of methylation protects the bacterial own DNA from the nucleophilic attack. By transferring methyl groups to adenine or cytosine residues of the DNA of its own it produces N6-methyladenine or 5-methylcytosine.
Once the methyl groups added to the DNA sequence the enzyme cannot identify their recognition sequence and hence it escapes the digestion process.
The recognition sequence of the REase are mostly the palindromic sequences, complimentary in both sense and antisense strand.
What are palindromic sequences?
The sequences of DNA in a sense strand from 5’ to 3’ and antisense strand from 3’ to 5’, are same, these types of DNA sequences are the palindromic sequences. See the figure,
The restriction endonuclease digestion system helps the bacterial own DNA from the phage invasion and from the other virus.
In vitro process of Restriction digestion
From the discovery of the REase, different REase are utilised for different types of applications. Due to different recognition sites, different REase cuts different sequences of the DNA.
REase = Restriction Endonuclease
Humans genome is very big, millions of recognition site are present for one particular type of REase. Hence cutting one specific sequence in a whole genome is nearly impossible by REase.
Read more on genome: What is genome
Take look at the different types of cutting sites of the REase:
Now let’s do restriction digestion.
The process of RE starts with DNA extraction. We have to extract the high quality of DNA from the source. The best method for DNA extraction for the restriction digestion is phenol-chloroform DNA extraction method, an enzymatic method of DNA extraction or you can use the ready to use DNA extraction kits.
Choose one of the methods of DNA extraction and extract DNA with ~1.80 purity having a good quantity of DNA.Different types of DNA extraction methods
Now choose the sequence of interest which you want to examine or digest. Take that sequence and design primer with the help of the primer 3 software. You can also follow our guide for designing primers. Read the article here,
Now cut your DNA sequence with the help of the NEB cutter software and select the REase which generates only 2 or three fragments with the length more than 70 bp.
Suppose you selected EcoR1 restriction enzyme which cuts the DNA at 5’-GAATTC-3’. Furthermore, this sequence is present once in our DNA of interest.
Now with the help of the appropriate PCR protocol amplify the DNA by the selected primers. This will generate millions of copies of the DNA fragment of our interest (having the restriction site for the EcoR1).
You can follow our guide for designing the PCR reaction. The standard PCR protocol is as followed. After the amplification, run the product on 2% agarose gel for confirming the amplification.
After that, design the restriction digestion protocol as per the manufacturer’s protocol or you can follow our protocol. It is as simple as discussed here,
|REase (EcoR1) (10 unit/μl)||1.0|
|10X buffer (REase buffer provided by the supplier)||3.0|
This protocol is for 30μl reaction and this reaction gives good results. So use 30μl reaction for the Restriction digestion.
Now, incubate the sample at 37℃ overnight or 60℃ for 1 o 2 hours (if the enzyme is fast digest) or follows the instruction given by the supplier. After the incubation, our restriction digestion is ready for loading on the gel.
Interestingly, the restriction digestion products might be too small hence it is difficult to resolve it on 2% gel, use 3% of agarose gel for the restriction digestion this will gives sharp and accurate DNA bands.
Read our article on how to do agarose gel electrophoresis: agarose gel electrophoresis
Factors affecting the restriction digestion:
pH of the buffer: pH is a very important factor in any of the genomic procedure. The pH of the reaction or the buffer must be between 7.0 to 8.0 which protects the DNA. Also, the buffer must contain the Tris-HCl.
Read more on Tris buffer: Tris-EDTA (TE) buffer in DNA extraction
Nuclease-free water: water is one of the important ingredients in restriction digestion procedure for balancing the ionic strength and the reaction. But, the water used into the restriction digestion must be nuclease-free, otherwise, other nucleases cut DNA randomly leads to false positive results.
The water must be de-ionised, double distilled, nuclease-free.
The requirement of Ions: the restriction endonuclease are the enzymes and as we know, ever enzyme required ion as a cofactor to enhance their activity. Mg2+ ions are the essential need in any types of restriction digestion, however, other ions such as Na+ and K+ are also needed into the reaction.
Nonetheless, all the essential ions are present in the buffer.
The temperature of the reaction: must be 37℃. All the enzymes are active at the body temperature hence all the digestion reaction must be carried out at 37℃ temperature, also the maximum activity of the enzymes are recorded at the temperature nearby ~60℃. We can also fast-digest the DNA at 60℃ to 65℃ temperature for 1 to 2 hours.
Other factors: the DNA sequence must be methylation-free, or the site of enzyme recognition must be free form the methylation otherwise the enzyme can not cut the DNA.
Remember, All the plastic wares and glassware used during the restriction digestion must be autoclaved, nuclease-free and contaminant free.
The DNA must be free from the contamination such as phenol, chloroform, DMSO and ethanol. These contaminants lead to star activity of the enzyme.
what is the star activity of endonuclease?
How to name restriction endonuclease?
Restriction digestion results analysis
Now, our digested DNA product is ready. We have to analyse the results whether the DNA is digested or not.
Suppose we have two different alleles for one particular gene having a point mutation. The point mutation is a site of REase recognition indicates that in normal wild type allele the restriction endonuclease will cut the DNA and in the other allele, mutant allele, the REase can not cut the DNA.
The results of the experiment are shown in the figure below,
Suppose our PCR product is of 300bp and the EcoRI creates two fragments after digestion (one with 70bp and one with 230bp).
Therefore, in the homozygous normal alleles, having the cutting site creates two different fragments after the digestion. Both the fragments are shown into lane two.
In the case of the homozygous mutant alleles, both alleles lack the recognition site due to the mutation from G to A, hence the EcoR1 can not cut it. The uncut allele with 300bp fragment is shown into lane 4.
In heterozygous genotype, one allele has the recognition site and one does not have it. Hence one allele is digested by the EcoR1 and one remains uncut. The three different bands with 70bp, 230bp and 300bp are shown into lane 3.
Also, the untreated PCR product is loaded into the 5th lane having the 300bp band of the DNA (as a control).
Suppose the normal allele in the population have the restriction digestion site, then in each case, the EcoR1 cuts the fragment into 70bp and 230bp, if remains uncut indicates the mutation. By doing this we can identify mutation at this site.
Methods of restriction digestion
The method what we are discussed into the earlier part of this article is RFLP, restriction fragment length polymorphism. The method is the combination of polymerisation and restriction digestion followed by the agarose gel electrophoresis.
The RFLP, AFLP and STRP are the most common types of methods used into the research as well as in diagnostic which contains the restriction digestion.Genetic markers
In recent time, the Restriction fragment length polymorphism is most popular in disease diagnosis of single gene disorders.
The AFLP, amplified fragment length polymorphism is also of the important tool used in the plant research. Also, the short tandem repeat polymorphism is best applicable along with restriction digestion.
Other methods which use restriction digestion is mapping and cloning. We will discuss the cloning and mapping in one separate article.
The restriction digestion is further, involve in the method of gene editing, especially, in the zinc finger nuclease method of gene transfer.
Direct digestion is also one of the best methods for the digestion of the whole genome, it is used to creates the library for microarray and DNA sequencing.
Read more on DNA sequencing
Application of restriction digestion
- Population-wide polymorphism and mutation study: The population wise study is very important for identification of the harmful alleles, the restriction digestion method is one of the powerful tools in studying the single gene mutation in different diseases such as sickle cell anaemia and Huntington’s disease. Also, new variations or polymorphism can be identified but can not be characterised
- Linkage mapping is possible by the restriction digestion method.
- Used in DNA probe preparation
- DNA fingerprinting and paternity verification is possible by the RFLP method of restriction digestion. Restriction digestion was commonly used in the criminal verification and paternity test since long.
- Cloning and expression vector preparation
The pathogenic mutant allele or new variations cannot be characterised by the restriction digestion which is the only limitation of it. For those who want to continue their carrier into genetics must learn the restriction digestion method. even if it is very basic, tradition and outdated techniques, in the era of gene transfer and DNA sequencing, one must learn traditional methods for gaining knowledge.
Since long and even in the future, the restriction digestion method remains gold standard for single gene disorder and point mutation. it is easy, cost effective and fast.