Restriction Mapping

Suppose you wish to visit a place you have never been before. However, the route is not known to you. What would you do? There are two options. The first option involves locating the place through an internet route map and finding out the best possible route if you intend to drive. The second option involves inquiring about a travel agency for the guidance. Knowing the route is important to reach a particular destination. A route map shows the exact location and the route to reach that place. Similarly, the genome map locates the position of a gene. Just like mapping the locations, it is possible to map the genes located on the chromosomes.

Image 1: Restriction mapping

Structural genomics is an important branch of genomics. It involves genetic and physical maps as guides in determining the gene location and establishing linkage studies. It involves neglection of repetitive DNA containing regions as they create unwanted results while sequencing. For handling large genomes, convenience lies in preferring the physical maps instead of genetic maps. Accuracy and resolution of physical maps make them the first choice. A plethora of physical mapping involves important techniques for not just generating a physical map but also to analyze the cloned DNA fragments. Three main techniques include restriction mapping, FISH, and STS mapping.  Analyzing the genes and cloned DNA sequences help in determining specific restriction site arrangement. Do you know what a restriction site could be? The word restriction indicates a specialty of a particular site. A restriction enzyme or an endonuclease recognizes specific sites and cuts there. These enzymes are known as molecular scissors. These enzymes break the DNA chain at a particular target nucleotide sequence such that it gets cleaved. Hence a site chosen by a restriction enzyme is known as a restriction site. Restriction digestion changes the size and number of the DNA fragments. Genetic engineering widely exploits this property of the restriction enzymes. Involving gene transcripts enhances the determination of the tissue specificity and the gene expression levels.

Restriction mapping is a physical mapping technique involving fragments of DNA separated by lengths marked in the number of bases. A restriction map is analogous to a linkage map. RFLPs or the restriction fragment length polymorphisms involve variations in DNA banding patterns of electrophoresed restriction digests from different individuals. RFLP mapping may help to locate polymorphic restriction sites. However, there is a limitation. The non-polymorphic sites are missed out and remain unmapped. Hence, the restriction mapping replaces the RFLP technique. The restriction mapping involves polymorphic and nonpolymorphic sites. Restriction enzymes cleave the genomic DNA into relatively smaller fragments. Using restriction maps involves many benefits. The restriction maps guide in cloning genes or cDNA.

Restriction mapping enables the determination of correct recombinant DNA molecules. Genome sequencing requires restriction maps in primitive stages.

Constructing a restriction map:

Suppose Eco RI and Bam HI restriction enzymes digest a DNA with a particular size. The resultant fragments obtained by using restriction endonucleases are known as restriction digests. Nomenclature of the restriction enzymes involves their source organism. For example, Eco RI is a restriction enzyme isolated from an E. coli RY13 strain. Bam HI is isolated from Bacillus amyloliquefaciens H. Eco RI and Bam HI consist of the specific recognition sequences and cleave at a particular position.

Enzyme name	Recognition sequence
Bam HI	5’-GGATCC-3’ 3’-CCTAGG-5’
Eco RI	5’-GAATTC-3’ 3’-CTTAAG-5’

Table: Enzyme and the recognition sites

While constructing a restriction map, it is necessary to use a proper concentration of the enzyme and follow the rule as per the conditions required for obtaining a restriction digest. The fragments obtained from restriction digestion are of predictable sizes. Suppose the DNA samples are digested with Eco RI and Bam HI. One more sample gets digested with a combination of Eco RI and Bam HI. Agarose gel electrophoresis separates the fragments as per their sizes. An electrophoretic apparatus consists of agarose gel with wells for loading the DNA samples. Five consecutive wells involve marker DNA, control sample, DNA digested with Eco RI, DNA digested with Bam HI and a digest of Eco RI+ Bam HI. The DNA fragments with short length migrate faster, thereby separating them as per the sizes. Cutting a DNA with both the enzymes is known as a double restriction. It enables mapping of three restriction sites. A large fragment consisting of two Bam HI sites again gets an enzyme treatment. Hence it synthesizes partially digested fragments with a few uncut sites. The separated DNA fragments in the gel get stained using ethidium bromide for visualizing the bands under ultraviolet light. Restriction mapping sometimes leads to fragments having the same sizes. Hence measuring such fragments is difficult. Two classes of rare cutters may be helpful. Some enzymes cut with seven to eight nucleotide sequences such as Sap I and Sgf I. Another class of enzymes involves recognition of 5’-CG-3’ sequence site. An example includes Sma I enzyme. Separation of Fragments larger than 50 Kb involves orthogonal field alteration gel electrophoresis (OFAGE).

Image 2: Steps involved in restriction mapping

Construction of plasmid checked by restriction mapping:

A confirmatory test to check the plasmid construction includes restriction mapping. Plasmid vectors carry clonal DNA segments. For example, Eco RI-Eco RI fragment gets inserted into a pUC 19 vector in two orientations. There is a restriction site known as Aat II site for Aat II restriction enzyme. A foreign DNA gets inserted into the vector pUC 19, which is a highly preferred vector in molecular biology. The concept of optical mapping came into the picture while cloning large DNA fragments in YAC and BAC vectors.

Gel stretching and molecular combing:

Optical mapping recruits microscopic examination of cut DNA molecules. The DNA gets attached to the slide without forming clumps. Gel stretching technique is a preparation of a gel stretched DNA. Following are the steps of gel stretching. First, a restriction enzyme is used to coat a clean grease free slide. The molten agarose containing chromosomal DNA is pipetted out on the slide. Solidification of the gel enables stretching of the DNA present on the slide. The reason behind stretching the DNA could be due to gelation. The addition of magnesium chloride activates the restriction enzymes which cuts the DNA molecule. The visibility of the gaps representing the cut sites is due to the coiling of the molecules. The molecular combing technique primarily includes dipping a coverslip into a DNA solution. The technique produces a comb of parallel molecules. The coverslip removal involves a constant speed of 0.3 mm/s. After drying the coverslip, the DNA molecules get retained as an array of parallel fibers. The immobilized DNA gets a restriction enzyme treatment and visualized using DAPI staining.

References:

[1] Molecular Biology, David P. Clark, Nanette J. Pazdernik
[2] Genetic Engineering, Verma P.S. & Agarwal V.K.
[3] Genomes, T.A. Brown

© Copyright, 2018 All Rights Reserved.

A Review on Restriction Enzymes

The enzymes involved in cleaving the specific regions in the genome are known as restriction enzymes. They are also known as molecular scissors. They are used widely in molecular biology. A restriction site is a specific region in the DNA consisting of bases cleaved by these enzymes. The process in which the restriction enzyme cleaves at a particular site is known as restriction digestion. The products in the form of the nucleic acid fragments are known as the restriction digests. They are known to play a crucial role in mapping, sequencing, and cloning. Most of the restriction enzymes belong to the class of endonucleases. It is an enzyme cleaving or hydrolyzing the phosphodiester bond within a DNA. The knife and fork model of DNA replication represents a triangular structure. This triangular structure is a sign of an endonuclease enzyme. The DNA molecule consists of internal phosphodiester bonds which get cleaved through restriction endonucleases.

In somatic tissue or cells, these enzymes are capable of hydrolyzing DNA by introducing double-strand breaks. Naturally occurring restriction endonucleases are known as the cell protectors. They protect the cells from viral infections. For research and analysis purpose, the extraction source of the enzymes involves microorganism or chemical synthesis.

Properties of restriction endonucleases:

A restriction enzyme recognizes a restriction site at 3’ carbon and phosphate group of a phosphodiester bond. The resultant fragments have 5’ phosphate group and 3’ hydroxyl group. Bacteria and green algae chlorella have naturally occurring restriction enzymes. Bacteria undergo a process of methylation and modify the restriction sites. Hence, the bacterial cells get protected from the action of restriction enzymes. Arbor, Nathans, and Smith received a Nobel prize in discovering the restriction enzymes. Many restrictions enzymes select sites having an axis of symmetry. The number of cuts made by restriction enzymes gets determined by the frequency of occurrence of the restriction sites. The restriction sites occur in a randomly distributed base pair. The formula (1/4)ⁿ indicates the probability of the occurrence of the restriction site with a 50% GC content. (Where n indicates the no. of nucleotide pairs in the recognition sequence).

Image: Restriction digestion (Treatment of the DNA sample with restriction endonucleases leads to the synthesis of fragments of different sizes.

Nomenclature:

The nomenclature of the restriction enzymes involves the organisms from which they were isolated. The first letter belongs to the genus and the second and third letter from the species. It involves an italic or underlined font followed by Roman numerals. For example, EcoRI is an example of a restriction enzyme obtained from E. coli strain RY13. Thus, ‘Eco’ belongs to E. coli, and RI belongs to the strain RY13. There are four classes of the restriction enzymes based on the recognition sequences.

Enzymes with no. of recognition sequences in base pairs	Property
4	Symmetrical sequences
6	Symmetrical sequences
8	Symmetrical sequences
spacer sequence	Non-specific sequences

Table: Enzymes and their properties 

Constructing a restriction map:

A restriction map is a kind of a physical map. It consists of a piece of DNA with restriction sites specific for endonucleases. The number of bases decides the sites of separation. Restriction enzymes create two types of ends such as sticky or blunt ends. A staggered cut in the restriction site with symmetrical nucleotide sequence generates sticky ends. There are two types of ends such as 5’ or 3’ overhanging ends. When a restriction enzyme cuts at a symmetrical nucleotide sequence of a restriction site between the two base pairs, the blunt ends get created. When the DNA gets digested with the restriction enzymes, and the resultant fragments get separated on a gel, the banding patterns are visible after staining. They are known as restriction fragment length polymorphisms (RFLPs). Sometimes the restriction sites vary in different individuals. A restriction enzyme cleavage site present in an individual may be absent in another individual. RFLPs help in mapping the genes or polymorphic sites. However, the non-polymorphic sites get missed. Restriction mapping solves this problem. A single restriction enzyme or combination of restriction enzymes cleave the DNA efficiently. Thus we obtain the fragments of different sizes measured in kilobases. The electrophoretic gel separates the fragments based on their sizes. Autoradiography helps to visualize the bands and cut the desired portion of the gel to take the DNA fragment.

Cloning Vectors:

A cloning procedure primarily uses a piece or a fragment of DNA obtained from the restriction digestion. The fragment of DNA gets inserted into the vector. The technique involves two main steps. First, the plasmid gets cleaved at a unique restriction site with an appropriate restriction enzyme. The second step involves the insertion of a piece of DNA cut with the same enzyme. The procedure further involves mixing of the cloning vector and a DNA fragment. It results into base pairing and annealing the two single-stranded ends. An enzyme known as DNA ligase acts as a sealing agent. It seals the gaps formed due to a phosphodiester bond.

Applications of restriction enzymes:

1.     DNA typing:

DNA fingerprinting or DNA typing determines the paternity and maternity of an individual. It helps in crime investigation. The establishment of variability in the ethnic groups involves DNA typing. Endangered species determination and genetic variability studies involve DNA typing techniques. Forensic medicine largely involves DNA fingerprinting techniques. Initially, the DNA obtained from the suspect individual gets a treatment with the restriction enzymes. The restriction digests obtained from the procedure include DNA fragments of different sizes. The fragments get separated as per their sizes in an electrophoretic apparatus. Using a Southern blot and a probe hybridization technique, visualization of the DNA bands of the suspect involves autoradiography.

It is not possible to separate the DNA fragments without the restriction enzymes. Hence, obtaining the information of the suspect invites difficulties. In the detection of paternity or maternity cases, the DNA obtained from the child and his alleged parents have to undergo restriction enzyme treatment. Autoradiogram shows a banding pattern of each sample as a DNA fingerprint.

2.     DNA molecular testing:

Genetic testing such as disease gene detection requires restriction enzyme treatment. The requirement of the restriction enzymes using PCR determines the presence of microsatellites. RFLP analysis and restriction mapping utilize the restriction enzymes. Detection of genes associated with diseases such as sickle cell anemia, phenylketonuria (PKU), and others become easy to detect.

3.     Gene Cloning:

The isolated DNA gets cleaved through RE treatment. The fragments obtained through RE treatment mix with the cloning vector and get integrated into the restriction sites in the cloning vectors. The cloning vector gets transformed into the host and allowed to replicate. The process is known as molecular cloning. Identical copies of DNA of interest obtained from the above process are known as clones. Expression of genes, synthesis of protein products, gene therapy, and many other important applications require gene cloning.

4.     Preparing recombinant vectors:

Restriction enzyme treatment targets multiple cloning sites or the restriction sites in a plasmid or any other vector. The fragment of interest gets inserted into the multiple cloning sites or a polylinker and gets transformed into the host. The host (bacterial cells) plated on a suitable medium show the growth of the recombinant colonies.

5.     Industrial applications:

It is possible to grow plant hybrids showing resistance to herbicides, pesticides, and drought tolerant varieties. Restriction digests obtained through restriction enzyme treatment get inserted into a vector. Then the DNA gets microinjected into the animal’s pronucleus to obtain transgenic animals. Hence, it is possible to produce a recombinant protein product using a transgenic animal. Example, the gene of interest expressed in a mammary tissue helps protein secretion in the milk.

6.     Genomic and cDNA libraries:

 The recombinant DNA libraries consist of a collection of clones with the desired DNA sequence. The fragments subjected to restriction enzymes get cloned into a vector to obtain a collection of clones known as genomic or cDNA libraries. 

References:
[1] Essential Genetics: A Genomics Perspective, Hartl, Elizabeth W. Jones
[2] Molecular Biology, David P. Clark, Nanette J. Pazdernik
[3] Genetic Engineering, Verma P.S. & Agarwal V.K.
[4] Molecular Biology and Genomics, Cornel Mulhardt

© Copyright, 2018 All Rights Reserved.