Screening of clones

Just like collecting books and arranging them in a library, the recombinant DNA library stores the cloned DNA fragments. For selecting a particular clone, we need to screen these libraries. Hence, before knowing the screening procedures, we must know the types of libraries. There are two main types of libraries such as cDNA library and genomic library. The cDNA library involves a collection of cloned cDNAs produced from a mRNA. The DNA sequence gets represented at least once in a genomic library.

1.   Screening a cDNA library:

For screening, we need to search for a cDNA clone encoding a protein. An antibody probe screens specific cDNA plasmid. It involves the use of an expression vector. Since we need to search for a cDNA clone encoding a protein, we use expression vectors. An expression vector is nothing but the vectors designed for the expression of the genes. When the cDNA gets cloned into an expression vector, the gene gets expressed and produces proteins. The cDNA gets inserted into an expression vector such that it gets fixed between the promoter and the termination signal. When cDNA gets inserted in the host, the gene gets expressed. The selection of the host involves E. coli cells. The expression vector containing cDNA gets inserted into the bacteria through the process of transformation. Why only expression vectors? An expression vector expresses the genes efficiently. Screening the cDNA clones requires a specific host such as E. coli. The growth of the cells helps us in identifying the recombinants and non-recombinants. It depends on the expression of the cDNA or the desired gene. Designing an expression vector depends on the type of organisms such as a prokaryote or a eukaryote. Hence, for every type of an organism, an expression vector design gets ready. Plus, we add a purification tag or a fusion partner along with the cDNA or a cloned gene. It helps in obtaining a purified product.

Plating the bacterial cells (transformed with the vector) on a selective medium allow the bacterial colonies to grow. Individual colonies get picked up and transferred to a microtiter dish. It is a 96 well dish. A microtiter well serves as a liquid holder. Several milliliters of liquid gets filled in the microtitre dish or well. Microtitre dish or a well has many applications. They serve in screening, filtration, separation, storage, culturing cells, detecting the anti-microbial activity, and many other uses. Microtiter dishes adjust with the temperatures. The evaporation of the solvent occurs more after heating.

Next step involves a pertiplate with a selective medium. A membrane filter placed on the Petri plate for getting the replicas of the clones. The selective medium is appropriate for the recombinant molecules. The pattern of the growth of colonies on the membrane filter is the same as the colonies growing on the microtiter wells. After observing or allowing growth of the colonies, the filter gets peeled from the dish. The cells get lysed using a lysis solution in situ. The protein product of cDNA gets bound to the filter. Next step involves incubation with an antibody labeled radioactively. The placement of the filter in the X-ray film helps in observing the expressed protein of interest. This process is also known as autoradiography. The development of X-ray film gives dark spots in the region where radioactive probe gets bound.

Image 1: Procedure for Screening of clones

Image 2: Antibody-Protein interaction

2.   Screening a genomic library:

The cloned gene coding for mRNA molecule gets identified using plasmid genomic libraries. The technique involves DNA probes. The construction of the genomic libraries depends on a vector such as a plasmid vector. The procedure for screening a genomic library is similar to that of the cDNA library. The host used in screening procedures involve E. coli bacterial cells. The first step involves the transformation of genomic libraries into E. coli cells. The cells plated on a selective medium help in obtaining the growth of the colonies. The technique involves replica plating. It helps in producing identical copies of a series of the bacterial colonies on Petri dishes. First, the Petri plate consisting of the bacterial colonies gets inverted. The surface of this dish gets pressed against a cylindrical block and with a velveteen cover. Hence, the bacterial colonies present on the Petri-plate get transferred to the velveteen. There is an extension to this procedure. The pressing of the velveteen consisting of the cells from the original colonies to a plate consisting of a selective medium with a membrane filter allows the cells to grow on the filter. After the cell growth, the membrane gets lifted and processed for cell lysing.

After the bacterial cell lysis, the DNA gets denatured into single-stranded structures. Then the DNA firmly binds to the filter. After placing the filter on a heat-stable bag, the cDNA probes get incubated with cDNA probes. The labeling of the probes with radioactive or a non-radioactive element involves separate steps. First, the DNA gets denatured by boiling. Next, a quick way of cooling it involves ice for producing the single-stranded molecules. The synthetically obtained primers get annealed to DNA. Next, pairing with the hexanucleotide primers takes place. They get elongated by Klenow fragment of DNA polymerase I using radioactively labeled precursors (dNTPs). An example includes P³² labels. A special DNA precursor molecule gets involved in the case of non-radioactive labeling. The precursor involves the use of the digoxigenin dUTP. It also involves usage of chemiluminescent substrates. The labeled DNA molecules get diffused over the filter. Complementary base pairing and hydrogen bonding form DNA-DNA hybrids. The formation of the hybrids occurs between the probe and the colony DNA. Washing the filter with a suitable solution helps to remove any unbound probes. Colonies on the film present as dark spots get detected using an autoradiography technique. Shuttle vectors and expression vectors also help in screening the genomic libraries.

Complementation of the mutations:

This test relies on the expression of the wild-type gene. Complementation test determines whether the two mutant sites are in the same gene. Crossing the two mutant phenotypes results in wild-type progeny leading to complementation. Let us consider an example of a yeast mutant lacking the arginine biosynthesis due to a mutant arg1 gene. The wild-type ARG1 gene synthesizes arginine. A designing a yeast-E coli shuttle vector involves the synthesis of the ARG1 gene product. The genomic library containing the ARG1 gene gets transformed into yeast containing the mutant phenotype, the ARG1 wild-type gene gets expressed. Hence, it results in complementation. Therefore, the ARG1 gene overcomes the functional defect of the arg1 mutant gene. The plasmid isolated from the cells helps in the characterization of the cloned gene.

Complementation test helps in identifying genes in the library. Shuttle vectors are bifunctional vectors. They grow and transform into prokaryotes as well as eukaryotes.

Heterologous and oligonucleotide probes:

A small and a defined nucleic acid (either DNA or RNA) identifying the specific molecules with complementary sequences are known as probes. Depending on the type of labeling, the probes matching their complementary sequences get detected through autoradiography. A probe searches a complementary sequence in a cDNA library, a genomic library, northern blot or in situ hybridization. Depending on a good degree of homology between the probes and the genes, the heterologous probes work. The probes consisting of equivalent genes obtained from other organisms are known as heterologous probes. Example, a mouse probe serves as a probe for a human genome library. It involves working with highly conserved genes. Hence, a heterologous probe is similar to the nucleic acid sequence but not complementary to the nucleic acid. Heterologous probing identifies related genes in the same organism. For example, wheat gliadin cDNA clones serve as probes. It hybridizes to a complementary sequence and a variety of other genes. Another example includes a yeast cytochrome c gene as a probe in identifying probable Neurospora cytochrome c clone in a Neurospora gene library.

Oligonucleotide probes identify genes or cDNA libraries. It is possible to clone genes that lack previous genetic information.

References:

[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2] Recombinant DNA Principles and Methodologies, James Greene

[3] IGenetics, Peter Russell

[4] Genetic Engineering, Verma P.S. & Agarwal V.K.

[5] Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh
[6] Gene cloning and DNA analysis, T.A. Brown

© Copyright, 2018 All Rights Reserved.

Recombinant library

A series of books in a library helps to study a suitable topic in detail. The detailed research and analysis of a topic require a lot of information. Different books cover a particular topic with different findings. Hence, the research studies involve reference to several books discussing a topic in different aspects. Hence, a book library is nothing but a collection of books discussing different subjects. Just like a book library, the molecular world depends on the DNA libraries. Recombinant DNA libraries consist of a collection of clones of individual DNA fragments, complementary DNA (cDNA) fragments, and chromosome fragments. Hence, they consist of three main types of libraries such as genomic libraries, cDNA libraries, and chromosome libraries. Recombinant DNA libraries help the researchers study a particular gene segment, DNA or a chromosomal segment isolated from any organism. It is possible to isolate a specific gene and study its structure, function, and expression using recombinant DNA libraries.

Clone libraries involve a collection of clones for generating mapping reagent. A collection of clones represent an entire genome. Hence, they supply individual clones of interest. Clone libraries contribute in physical and gene mapping techniques as well. Preparation of a clone library either involves a genomic DNA or a specific chromosome.

Image: Recombinant vectors

Genomic library:

They consist of at least one copy of every DNA sequence in the genome. It is possible to isolate and study a particular gene using a genomic library. The first step of constructing a genomic library involves digesting the DNA sample with restriction enzymes. Next step follows cloning the DNA fragment obtained from restriction digestion into a vector. The vector consists of a specific restriction site. First, the restriction site in the vector undergoes cleavage due to the action of a specific restriction endonuclease. The vector now has a space for new DNA insert. Upon treating the vector with new DNA fragment and specific ligating enzymes, the DNA gets inserted into the vector. Hence, the genomic library gets prepared. However, a limitation exists to the above method. Suppose, if a gene consists of one or more restriction sites for a specific restriction enzyme, it gets cleaved into two or more fragments smaller in sizes. In this way, it produces a large number of fragments. Cloning the longer DNA fragments into a vector helps in solving the problem. Mechanical shearing gives longer DNA fragments. Another approach involves partial digestion. The restriction enzymes recognize four to six base pair nucleotide sequences. Partial digestion results in the formation of overlapping fragments.

Uses of genomic libraries:

1.     Genomic libraries help in the sequencing projects such as whole genome sequencing, human genome sequencing, and sequencing related to another organism.

2.     It is possible to create many recombinant DNA molecules using a genomic library.

3.     A genomic library has a larger size. It consists of an entire genome of an organism.

4.     The genomic library helps in molecular cloning of a gene.

The cDNA libraries:

The mRNA molecules help in deriving the cDNA (complementary DNA). Cloning the cDNA molecules involve a contribution of a suitable vector for producing cDNA library. Most f the mRNA molecule consists of a poly(A) tail. Polyadenylation process occurs after the mRNA synthesis and capping. The poly (A) tail with mRNA gets purified with the help of deoxythymidylic acid known as oligo (dT) chain. The poly (A) tails base pair with the oligo (dT) chains. The mRNA gets captured on the column leaving the remaining molecules. After annealing the oligo (dT) primer to a poly (A) tail, the primer gets extended using a reverse transcriptase enzyme. This enzyme helps in making a DNA copy of the mRNA strand. The second DNA fragment gets synthesized with the help of RNase H, DNA polymerase I, and DNA ligase. The DNA polymerase I synthesize a new DNA strand and removes the primers. Then the ligating enzyme joins them. The result is a double-stranded DNA which is known as cDNA or a complementary DNA.

Cloning of a cDNA involves a restriction site linker. It is a short, double-stranded piece of DNA with 8-12 nucleotide pairs in length. The linker has blunt ends. Hence, cDNA gets ligated with a linker (such as Bam HI) using a T4 ligase enzyme. Now the linkers get cleaved using a Bam HI restriction enzyme. The restriction enzyme cleaves the linker at a particular nucleotide resulting in sticky ends. The resultant product gets inserted into a vector cleaved with Bam HI. The recombinant vector gets transformed into a host such as E coli. There is a drawback of using a linker. The cDNA may have restriction sites just like the linker DNA. Hence, there are chances of cDNA getting cleaved with a restriction enzyme. It is required to prevent the cDNA digestion with the restriction enzymes. An adapter helps in doing so. Upon ligation, the blunt end of the adapter covalently attaches to the blunt end of the cDNA. It leaves a 5’ overhang at each end capable of base pairing with a vector.

Uses of a cDNA library:

1.     The cDNA libraries are small and precise. They do not involve the non-coding regions or the introns. Hence, they contribute to studying the coding regions or exons.

2.     They express eukaryotic genes in prokaryotes.

3.     Helps to study reverse genetics.

Chromosome libraries:

Libraries for specific human chromosomes are also available. Also, the artificial chromosome libraries are available. Examples include BACs and YACs. The Bacterial artificial chromosome (BACs) libraries consist of bacterial chromosomes or DNA cloned into vectors. YACs are also known as yeast artificial chromosomes and derived from yeast.

Deriving a human chromosome library involves separating the chromosomes by flow cytometry. The first step involves carefully breaking apart the dividing cells for obtaining the chromosomes. The next step involves staining the chromosomes with a fluorescent dye. The larger chromosomes require more dye and fluoresce more brightly. Chromosome libraries serve in STS mapping. We use chromosome library as a mapping reagent or obtain a DNA sequence using overlapping clones. 

References:

[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2] Recombinant DNA Principles and Methodologies, James Greene

[3] Gene Cloning and Manipulation, Christopher Howe

[4] Genetic Engineering, Verma P.S. & Agarwal V.K.

[5] Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh
[6] Gene cloning and DNA analysis, T.A. Brown

© Copyright, 2018 All Rights Reserved.

A Review on Gene Cloning

Suppose you have generated important data and you do not want to lose it. What would you do to protect it? You may either file it or store it in a folder. Or you would generate multiple copies of the information using a Xerox. The Xerox machine reads the information in your paper and gives you a printout in the form of a photocopy. Thus, Xerox creates exact replicas of the document. Similarly, our body protects the genetic material by generating multiple copies. An example involves the replication process.
Scientists felt the need to generate multiple DNA copies. The process of cloning involves obtaining multiple copies of a gene or a DNA segment using technology. Each identical copy of a DNA segment or a gene segment is known as a clone. Human genome project utilizes maximum amounts of clones in various experiments. Many such cloned DNA collections make up a clone library. Cloning is not only applicable to DNA or a gene segment but also possible with the entire cell. This type of cloning exploits the natural process of cell division to make many copies of an entire cell. The genetic makeup of the cloned cells is known as cell lines. They are identical to the original cell. Gene cloning involves recombining a population of DNA molecules into the vector. The recombined DNA molecules contain the gene of interest. The DNA is inserted into a vector so that each vector consists of single DNA from the original population. The screening of the colonies revealed the presence or absence of the DNA of interest. Thus, understanding cloning requires knowledge of the clones and their production.

Image 1: Gene Cloning

The review article discusses various aspects of cloning:

1.     Restriction enzymes and cloning vectors

2.     Collection of clones in the form of a library

3.     Screening of specific clones

4.     Analyzing cloned DNA through a molecular approach

5.     Identifying adjacent clones

6.     Applications

Cloning involves producing multiple copies of the genes or DNA segment of interest. In short, the procedure involves digesting a DNA segment with restriction enzymes, introducing it in a vector, transforming the recombinant vector into the host and observing the expression of the gene of interest. The screened clones are later on analyzed using molecular techniques.

Restriction digestion:

The restriction enzymes are used to digest the DNA. These enzymes are endonucleases capable of cleaving specific sites in the DNA known as the restriction sites. The restriction sites have specific nucleotide base pairs. A restriction enzyme cleaves a specific base by breaking the phosphodiester bond. Cleaving of the DNA can be achieved using one or a combination of restriction enzymes. The process of cleaving the DNA to obtain fragments of different sizes is known as restriction digestion. Separation of the DNA fragments is possible with the electrophoretic technique. Smaller fragments travel faster than the larger fragments, thereby forming bands. After visualizing bands, the portion of the gel consisting of a DNA fragment is cut and used for cloning experiments. Here are the two options. One of the options is to make multiple copies of the gene using amplification techniques such as PCR. The second option is to incorporate the DNA into the cloning vector. PCR or polymerase chain reaction is a process of amplifying the gene or a DNA using a sophisticated thermal cycler. Cloning DNA using a vector involves inserting the desired DNA into a plasmid or another vector.

Cloning vectors:

A cloning vector replicates within the host organism. It consists of a restriction site which can be cleaved for inserting the desired DNA fragment. Various cloning vectors are available. A plasmid vector is a plasmid or an extrachromosomal material of the bacteria. It is isolated and used as a vector. It consists of three sites such as the origin of replication, a selectable marker gene, and one or more restriction sites. For example, pUC 19 vector has a polylinker site or multiple cloning sites. On treating the restriction site with a restriction enzyme, that particular site gets cleaved and the desired fragment of DNA gets inserted in that place. Other examples of vectors include shuttle vectors, expression vectors, cosmids, phasmids, and artificial chromosomes. Shuttle vectors are bifunctional. They are capable of insertion into two or more hosts. Expression vectors express the desired gene to get the protein product.

Clone Library:

It can be anything from a collection of clones including cDNAs, chromosomes, and genomes. A cDNA library is nothing but a collection of cloned cDNA sequences synthesized from mRNA. A collection of cloned DNA in which the sequence appears at least once is known as a genomic library. The clone libraries appear analogous to book library. Just as the books provide plenty of information, the clone libraries provide a lot of information about the genome. Genome libraries production involves inserting the required DNA digest into a vector and storing the product. However, there are other methods too. Gene splitting with a restriction enzyme sometimes gives the undesired product. Such cases involve other techniques like mechanical shearing or partial digestion. Large DNA insertion involves mechanical shearing. The process of screening is made easy using chromosome libraries. 24 different human chromosomes libraries are available. The preparation of the cDNA libraries involves mRNA molecules as raw materials. After making clone libraries, a specific gene is found using screening techniques.

Image 2: Transformation of the recombinant plasmid

Screening procedures:

The screening of cDNA libraries involves an antibody probe. The first step is to transform the recombinant plasmid consisting of the required cloned cDNA into a host such as E. coli bacteria. Next step involves plating the bacteria on a selective medium to observe the growth of the colonies. Transferring the colonies into a microtiter well enables the bacteria to grow. The transfer of the colonies to the membrane filter also enables them to grow to get the expression of the gene. Removal of the filter and cell lysis enables the protein product exposure. Radioactively labeled antibody treatment with the protein product is the final step. Autoradiography helps the observation of clones in the form of dark spots. Screening a genomic library involves a similar system. It involves plating the transformed bacteria on a medium and processing them through replica plating. Here, the probed DNA is used instead of a radioactively labeled antibody. Complementation test helps to identify the specific genes.

Analyzing cloned DNA:

Analysis of clones involves molecular biology techniques such as restriction mapping, southern and northern blotting. Restriction mapping includes the physical mapping of the genes. Restriction digestion gives rise to DNA fragments of different sizes. Electrophoresis separates the fragments in the form of bands. Southern blotting uses a nitrocellulose membrane filter placed on the gel immersed in the buffer solution. Capillary action of the blotting paper enables the DNA to come in contact with the buffer and transfer to the membrane filter. The filter gets exposed to probes which hybridize with the DNA. Autoradiography helps to detect the DNA. Southern blotting mainly involves blotting DNA. Northern blotting involves blotting RNA instead of DNA.

Adjacent clone identification:

Genes between the flanking markers are found through chromosome walking. Hence, adjacent clone identification requires chromosome walking. The overlapping clones help to study larger DNA segments.

Applications of cloning:

1.     Recombinant DNA technology or genetic engineering: Cloning techniques involve obtaining large amounts of pure DNA. Expression of the desired products including proteins, vitamins, enzymes, growth factors, and other biomolecules is possible with recombinant DNA technology. Cloned genes help to detect mutations. Plant vectors consisting of the desired gene improve the crop quality. Cloning enables development of transgenic plant varieties with stress tolerance, drought tolerance, and herbicide or pesticide resistance. Particular protein synthesis is achieved using expression vectors.

2.     Transgenics: These organisms, plants, animals or microbes, involve manipulated genes cloned into several copies. The genes of interest express phenotypically. Examples include knockout animals.

3.     Gene therapy: It helps in replacing the gene that has lost its function. The defective gene identification accompanies cloning the normal gene inserted into the affected individual through a vector. The first gene therapy was successful in children suffering from ADA deficiency. The gene therapy is a new way to treat genetic disease because the abnormal gene is corrected or replaced.

4.     In vitro fertilization: Microsurgery involves removal of the nucleus from the donor’s oocyte. The microinjection of the surrogate mother’s somatic cell nucleus into the donor’s oocyte makes the cell chimeric. The chimeric cell cultured in the laboratory develops into an embryo. The embryo gets implanted into the surrogate mother’s uterus.

     References:
[1] Gene cloning and DNA analysis, T.A. Brown
[2] Recombinant DNA Technology, Sardul Singh Sandhu
[3]Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh
[4] Gene Cloning and Manipulation, Christopher Howe

   

© Copyright, 2018 All Rights Reserved.

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.