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.

Bacteriophage gene mapping

Viruses replicating themselves in the bacterial cells are known as phages. They are capable of surviving through a different mechanism. Their genes are easy to study since these entities are totally dependent on the bacteria. Plus they naturally exhibit the property of recombination of genetic material. Thus bacteriophage gene mapping is an interesting topic for geneticists. These phages are vectors since they are capable of transferring the genetic material from one bacterial cell to another bacterial cell. They are model organisms used in biological research. A gene map is a linear designation of mutant sites within a gene based on intragenic recombination. It could exhibit a sequence with regulatory sites, introns, exons and other elements. The principles used in eukaryotic gene mapping are applicable for mapping phage genes. Two main principles follow this. The genetic material gets exchanged between strains differing in the genetic markers. The second principle involves the detection of recombinants and counting them.

The intragenic or intergenic recombinants are mapped separately. These methods involve mapping the mutation sites. The unit of mutation and recombination is a DNA base pair. Mapping of bacteriophage genes requires the knowledge of the phenotypes. Several mutations give differences in plaque appearance. Consider two phage strains differing in genotypes. One phage has a genotype h⁺r. It is wild-type for a host range. The mutants have an hr⁺ genotype. The phage with a mutant allele produces a clear plaque. The phages with the wild-type allele produce cloudy plaques. A genetic cross is performed to map these two genes. Two E. coli strains are strain B and B/2. First, the strain B bacteria are coinfected with two bacteriophages named h⁺r and hr⁺. The phage chromosomes replicate within the bacterial cells and undergo recombination. Then the cell lysis releases the progeny phages along with the recombinant phages.

Fine structure and deletion mapping techniques were done using bacteriophage genes. The review focuses on both the mapping techniques.

Fine structure analysis:

Image 1: Fine structure mapping

Mutation studies in Drosophila led to the conclusion that the gene was sub-divisible by mutation and recombination. C.P. Oliver studied and proposed this concept in 1940

Intergenic mapping involves mapping the distance between mutational sites in different genes. Intragenic mapping involves mapping mutational sites within the same gene. Seymour Benzer worked on fine details of phage T4 genes. Fine structure mapping involved details within the gene. He worked on rII mutants of T4 phages. The rII mutants possess two main properties such as distinct plaque morphology and host range properties. When the r+ wild-type phages infected the E. coli bacteria plated on a solid medium, small turbid plaques with fuzzy edges appeared. The rII mutants produced large and clear plaques. There is a difference between wild-type and mutant strains. Wild-type r+ strains grow and lyse strain B or K12(λ). The rII mutants grow only on strain B. Thus the experimenter set out to construct a fine structure map of rII region using E. coli strain B as the permissive host. The experiment involved 60 independently isolated rII mutants. Different mutations formed the basis of crosses.

The rIIx and rIIy mutants were allowed to cross. Different mutations were written x and y respectively. Four types of progeny were the parental type such as rIIx, rIIy, four double mutants rIIx,y with r+ wild-type. The relative frequencies of the parental and the recombinants depended on the distance between the two alleles. The phage progeny were plated on E. coli B strain to calculate the total number of phages per milliliter.

Recombination frequency for two alleles

= (2 x No. of r+ recombinants x 100%)/ Total number of progeny

The number 2 indicates the double recombinants. The chances of reversions were lower than the smallest recombination frequency. Homoallelic and heteroallelic mutations were involved. Homoallelic mutations involved changes in the same nucleotide base pair within a gene. Heteroallelic mutations involved changes in different nucleotide pairs. The phage T4 gene map is about 1500 map units.

 Deletion mapping was used to localize unknown mutations. Maximum mutants isolated from the experiment were known as point mutants. The point mutations can revert. However, some rII mutants did not revert. Nor did they produce any recombinants. Such mutants were known as deletion mutants. Such mutants had lost a segment of DNA. The crosses performed between the mutants were analyzed. Seven standard deletion mutants involved in the cross. A1 to A6 and segment B included a total of seven segments. Thus, deletion mapping involves overlapping deletions to localize the position of an unknown gene on a chromosome or a linkage map. Gene structure analysis and mapping revealed that genes are units of mutations, recombination, and function. Determining the number of genes involved a test known as complementation test. The gene mapping studies and fine structure analysis revealed that the unit of mutation and recombination are the same. The base pair in DNA is the main criteria for determining both the factors.

Complementation test:

Image 2: Complementation test: The first Petri-plate shows the plaque formation. The second Petri-plate has no plaques. Thus, it helps in finding the complementation.

The test was used to find out whether the two different mutants belonged to the same gene. It is a mating test that determines whether the two different recessive mutations on opposite chromosomes of a diploid or a partial diploid will not complement each other or have a mutant phenotype. However, the same two recessive mutations on the same chromosome may show wild-type phenotype. Thus, complementation test is a test for allelism. Let us consider two cases for our understanding.

In case I, bacterium E. coli K12(λ) is infected with two phages with rIIA mutation and rIIB mutation respectively. Both the mutants make non-functional A and B products respectively. In this case, complementation occurs because rIIA mutant makes functional B product and rIIB mutant makes functional A product. Thus, progeny phages result in the formation of plaques.

In case II, bacterium E. coli K12 (λ) is infected with two phages having mutations in the rIIA gene. Hence, a non-functional A product and a functional B product were synthesized. In this case, complementation does not occur. Phage propagation does not occur due to lack of functional A product. Hence no progeny phages are produced. Thus, no plaques were visible on the bacterial lawn. These studies are inclusive of fine structure mapping and deletion mapping. A complementation map is a diagrammatic representation of the complementation pattern of mutants. Two types of lines drawn in a complementation map include overlapping and non-overlapping lines. The overlapping lines indicate non-complementary mutants whereas non-overlapping lines represent mutually complementing mutants. A complementation map is linear and may show lesions.

References:

[1] Microbial Genetics, Keya Chaudhari

[2] Molecular Genetics of Bacteria, Jeremy W. Dale, Simon F. Park

[3] Genetics, G. Ivor Hickey

[4] IGenetics, Peter Russell

[5] Genomes, T.A. Brown

© Copyright, 2018 All Rights Reserved.