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).
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| Image 2: Steps involved in 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
[2] Genetic Engineering, Verma P.S. & Agarwal V.K.
[3] Genomes, T.A. Brown
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