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Life Sciences For NET & SET Exams. Of UGC-CSIR

Section B and C
Volume-28 Contents

13. METHODS IN BIOLOGY A. MOLECULAR BIOLOGY AND RECOMBINANT DNA-METHODS 1

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*MUDRA* 13. METHODS IN BIOLOGY

Life Sciences For NET & SET Exams. Of UGC-CSIR

A. MOLECULAR BIOLOGY AND RECOMBINANT DNA METHODS ISOLATION OF PLANT DNA Isolating Genes The knowledge of gene isolation was developed after gaining the concept of physical and chemical characteristics of described DNA fragments- their sizes, shapes, and conformation can aid in the selection of methods used to isolate and purify those segments. Plants provide several special challenges for researchers interested in recombinant DNA research. No other class of living organisms has three separate genomes to analyze- a nuclear genome containing high molecular weight linear chromosomes, a circular mitochondrial genome, and a circular chloroplast genome. Intact, high molecular weight (>150 kbp) plant nuclear gDNA is used to construct genomic DNA libraries and to probe the plant genome for the presence of DNA markers, such as randomly amplified polymorphic DNAs (RAPDs) and restriction fragment length polymorphisms (RFLPs). Chloroplast cpDNA and mitochondrial m DNA, which are thought to incur fewer structural changes over time, are often used to study plant systematics. DNA is very easily damaged by shear forces; even rapid stirring of solution can break even with high DNA molecular weight into much shorter fragments. Consequently, DNA is recovered from cells by gentlest possible method of rupture, in the presence of EDTA to chelate the Mg2+ ions needed for DNase activity. Cell walls, if present, are digested enzymatically (e.g. lysozyme treatment of bacteria) and the cell membrane is solubilized using detergent. Cell disruption is performed at 4C using autoclaved glassware and solutions to destroy DNase activity. After release of nucleic acids from the cells, RNA can be removed by treatment with ribonuclease (RNase). The other major contaminant, protein, is removed by shaking the solution gently with water-saturated phenol, or with phenol/chloroform mixture, either of which will denature protein but not nucleic acids. Centrifugation of the emulsion formed by this mixture produces a lower, organic phase, separated from the upper aqueous phase by an interface of denatured proteins. The aqueous solution is recovered and deproteinized repeatedly, until no matter is seen at interface. Finally, the deproteinized DNA preparation is mixed with ethanol and allowed to precipitate out in freezer. After centrifugation the DNA pallet is redissolved in a buffer containing EDTA for protection against DNases and this solution can be stored at 4C for _____________________________________________________________________________ 2 Section B & C Vol-28

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at least a month. DNA solutions can be stored frozen, but repeated freezing and thawing tends to damage long molecules by shearing; so preparation in frequent use are normally stored at 4C. This procedure is suitable for total cellular DNA. If the DNA from specific organelle or viral particle is needed, it is best to isolate the organelle or virus before extracting its DNA. Now we will discuss several basic techniques related with gene isolation and identification.
Plant Tissue Preparation of Nuclei Protinase-K Treatment Phenol Extraction Isoamylalcohol : Chloroform Extraction Dissolve the Precopitate RNAse Treatment Phenol Extraction Asoamyl alcohol : Chloroform Extraction DNA precipitation with Ethanol

Fig.1: Major steps involved in DNA isolation

Isolating the DNA (Gene) for Interest For isolation of DNA from linear chromosomes of living organisms like plants, animals or from simple bacteria, it is necessary to break the cell for release of DNA which may be done by physical (homogenization or pressure), or biochemical means like solubilization with detergents or enzymes. The selection of enzyme for this process is dependent on the composition of the particular cells that are used. For example cellulases and proteases are used for plant materials, for fungal sample, chitinase is used. Lysozyme is commonly used for both bacterial and animal tissue samples. The DNA so obtained is then separated from the cell walls, membranes, and other cellular debris by means of centrifugation, often followed by density gradient centrifugation. The localization of DNAs in the gradient may be done by instrumental method (absorption at 260 nm or visually by means of fluorescence). The isolation of DNA is done by inserting a syringe into the side of the soft plastic centrifuge tube and withdrawing the bands visible in UV radiation. The isolated DNA may be further purified and physically characterized (for example, for size) by _____________________________________________________________________________ 3 Section B & C Vol-28

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means of electrophoresis in gel made up of agarose or of acrylamide. Isolation of genes is the in vitro gene manipulation as it gives us the ability to bypass the multiplicity of mechanisms that restrict gene transfer between unrelated organisms and are far more accurate and subtle in nature. The broad outlines of this technique are: isolation of gene of interest, insertion of a gene into an appropriate vector, transfer of foreign DNA (gene) through the vector into an appropriate host organism to-produce recombinant DNA and identification of recombinants. Methods of Isolation Foreign DNA (gene) of interest may be viral, bacterial or of plant or animal origin, therefore, different methods have been described: 1. Fragmentation by Mechanical Shearing: Random fragments of DNA can be generated by mechanical shearing. The desired fragments obtained by this method are without cohesive ends, therefore, the ligation (joining) with the vector can be facilitated with a process known as homopolymer tailing. For example, a tail of dC residues (deoxynucleotide triphosphate) can be added to 3-OH terminus of DNA of sheared fragment and a tail of dG residues to vector. The fragments to be cloned can then be joined to the reactor by annealing the tails. 2. Shot-gun method: There is a restriction enzyme which is specific for a six-base sequence of DNA. It is used to cut foreign DNA to get a piece of interest. One can obtained fragments of 3 to 4 genes by this method, if distribution of bases is random. The fragments obtained can be too small or too big if the number of cutting sites recognized by the enzyme used are too frequent or too sparse in distribution. It is also important that this cutting site falls on either end of the gene of interest and not in between. When the same enzyme is used to cut the vector DNA, cohesive ends are created, in most cases depending on the nature of enzyme, the joining of vector and the isolated DNA is possible. 3. cDNA Method: If animal gene is to be expressed in a bacterium or yeast, a suitable method is to isolate the messenger RNA (mRNA), first, concerned with the specific protein production. It is often impossible to express animal gene directly in bacteria because of the introns within the gene. The protein encoding parts of the genes are exons. Mature mRNA molecules in animal cells do not contain sequences complementary to introns as they are removed by processing. Introns are the sequences in DNAs which are in fact transcribed into mRNAs but are subsequently spilt out and therefore form no permanent constituents of the _____________________________________________________________________________ 4 Section B & C Vol-28