Preface This practical manual on molecular diagnostics is written to help the students and faculties who are doing practical of molecular biology, biochemistry and biotechnology. This practical manual covers the syllabus of ICAR as per 5th Dean Committee for B.Sc. Agriculture students. This practical manual can also be used by the students of under graduate and post graduate of other streams of biological sciences. This manual explains step wise laboratory practical procedure for different experiments. This manual will also be helpful for those students and researchers who want to plan the experiment in molecular biology laboratory. In the manual several actual figures of results have been given for example. From these figures the students, researchers and faculty members can compare their observed results. Total seventeen different experiments have been explained including screening of abiotic stress tolerant & sensitive genotypes, isolation & quantification of protein, isolation & assay of Reactive Oxygen Species (ROS) enzymes, separation of isozymes & protein by Polyacrylamide Gel Electrophoresis (PAGE), DNA & RNA isolation, quantification and quality test by spectrophotometer and agarsoe gel electrophoresis, PCR, RAPD, southern blotting and ELISA technique. The authors are also engaged in research and teaching in this area and having wide experience in molecular biology. In the end of each experiment some questions have been asked to solve by the students that will help in better understanding of the topic. This manual also contains set of objective questions related to the molecular diagnostics that will be very useful for their competitive exams and regular theoretical exams as well. The molecular biology techniques are gaining much importance in life sciences and without this now a day's life science experiments cannot be thought. The suggestions and critics for further up gradation of this manual is always welcome. We would like to thank to the publisher to bring this book in its final shape for publication.

Objective: To identify drought tolerant genotypes by PEG method in laboratory. Background information: Among different abiotic stresses, drought is considered to be most detrimental for agricultural production. A drought is a period of below-average precipitation in a given climatic region that results in prolonged shortages in water availability, whether atmospheric, surface water or ground water. A drought can last for several months or years. Drought has substantial impact on ecosystem and agricultural productivity. Some plant species are better able to tolerate drought stress than others. Genetic variability for drought tolerance also exists within many plant species (Blum, et al. 1980). The efficient exploitation of that variability for crop improvement would be facilitated by a fast and accurate screening method for identifying drought tolerant genotypes (Clark and Mc Craig, 1982). Several methods utilizing seedlings to screen for drought tolerance have been developed using an osmoticum such as polyethylene glycol (PEG) (Singh, et al. 1984). The other synonyms of PEG are Poly ethylene oxide (PEO) and Poly oxyethylene (POE); these other chemicals depend on the molecular weight. Chemical structure of PEG may be written as H”(O”CH2”CH2)n”OH (Kahovec, et al. 2002). PEG is used in certain abiotic stress experiments to produce high degree of osmotic pressure, normally in the order of 'tens of atmosphere' PEG produces osmotic pressure due to its flexible nature and water soluble polymer. PEG also makes certain specific interactions with bio-molecules. Due to above properties PEG is used as a specific chemical to produce osmotic pressure in several biochemical and bio-membrane related experiments. For some genotypes, the seedling response apparently is a good indicator of the plant's reaction to drought stress later in its life cycle. However, early screening of seed plants via seedlings tests must still be considered as a method for identifying genotypes that will tolerate drought stress in the vegetative stage (Andersen, et al. 1987).

Objective: To identify salt tolerant genotypes by Glycine Betaine (GB) method in laboratory. Background information: Salt or salinity is a biggest danger that causes reduction in crop productivity worldwide. Salinity reduces the germination percentage, plant vigour and ultimately loss in final crop yield. There are different physiological parameters that are affected by high level of salinity viz., oxidative stress, physical alteration of bio-membrane, nutritional imbalance, loss of cell division & cell expansion, water stress, genotoxicity and ion toxicity. Together, these effects reduce plant growth, development and survival. During the onset and development of salt stress within a plant, all the major processes such as photosynthesis, protein synthesis and energy and lipid metabolism are affected. During initial exposure to salinity, plants experience water stress, which in turn reduces leaf expansion (Petronia, et al. 2011). The water-soluble compounds which, are low in molecular weight, accumulate at certain concentration known as “compatible solutes” or “osmolytes” is the common strategy adopted by many organisms to combat the abiotic stresses. Some common compatible solutes are betaines, sugars (mannitol, sorbitol and trehalose), polyols, polyamines and amino acid (proline). Their accumulation is favored under water-deficit or salt stress as they provide stress tolerance to cell without interfering cellular machinery. The tolerant or sensitive species show difference in the stress tolerance level based on levels of accumulation of these compounds during abiotic stress condition. Accumulation of osmolytes like glycinebetaine (GB) in cells is known to protect organisms against abiotic stresses via osmoregulation or osmoprotection. Other roles of GB like cellular macromolecule protection and ROS detoxification have been suggested as mechanisms responsible for abiotic stress tolerance. In addition, GB also influences expression of several endogenous genes in plants.

Objective: To estimate total protein in the plant sample by Folin-Lowry's method. Background information: Protein assay can be done to determine the level of protein in solution by the Folin-Lowry biochemical method. The concentration of protein leads to change in color of the solution and based on different color in different protein concentration solution the change in color may be digitized by colorimeter or spectrophotometer. This technique was developed by Oliver H. Lowry in the year 1940s. The tyrosine and trytophan amino acid residues in a protein sample produces purple or blue color in the absorption maxima at 660 nm by using Folin-Ciocalteau (having phosphate and sodium tungstate molybdate) chemical reagent. The color intensity of protein sample depends on the concentration of above aromatic amino acids (tyrosine and tryptophan), hence every sample will vary for color. For protein estimation the BSA (Bovine serum albumin) is widely used as standard protein molecule due to its cost effectiveness, purity and easy availability. This method of protein estimation is very sensitive and can detect up to 10 μg/ml. This is a relative method of protein estimation and may be interfere by salts, Tris buffer, nonionic & cationic detergents, EDTA, lipids and carbohydrates. For reproducible assay the incubation time, pH (9-10.5) is critical in this method (Lowry, et al. 1951).

Objective: To separate protein fragments by using SDS-PAGE method. Background information Electrophoresis is the motion of dispersed particles relative to a fluid under the effect of limited uniform field of electricity (Dukhin, and Derjaguin, 1974). Ferdin and Frederic Reuss (Reuss, 1809) first time observed such phenomena of electrokinetic nature in 1807. They found that the clay particles move in the water by giving constant supply of current. It happened due to interface of charged particles surface and the surrounding media. The technique is used widely in chemistry, biochemistry and molecular biology to separate different molecules based on difference in charge, binding affinity and size. Electrophoresis may be of two types viz., Anaphoresis and Cataphoresis. Anaphoresis is the electrophoresis of anions (-ve charged ions) while, Cataphoresis is electrophoresis of cations (+ve charged ions). Electrophoresis is a major analytical technique in biochemistry that is used for separation of macromolecules like protein, enzymes and nucleic acids. Electrophoresis is a technique used in laboratories in order to separate macromolecules based on size. PAGE is abbreviated for Polyacrylamide gel electrophoresis that describes a technique widely used in biochemistry, forensics, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Mobility is a function of the length, conformation and charge of the molecule.

Objective: To extract and perform enzyme assay of polyphenol oxidase from plant. Background information: Polyphenol oxidase (PPO or monophenol monooxygenase or polyphenol oxidase I, chloroplastic) is a tetramer that contains four atoms of copper per molecule, and binding sites for two aromatic compounds and oxygen (www.worthington-biochem.com). The enzyme catalyses the o-hydroxylation of monophenol molecules in which the benzene ring contains a single hydroxyl substituent to o-diphenols (phenol molecules containing two hydroxyl substituents). It can also further catalyse the oxidation of o-diphenols to produce o-quinones. PPO causes the rapid polymerization of o-quinones to produce black, brown or red pigments (polyphenols) that cause fruit browning. The amino acid tyrosine contains a single phenolic ring that may be oxidised by the action of PPOs to form o-quinone. Hence, PPOs are some time referred as tyrosinases (Mayer, 2006). Jolley et al. (1974) referred to PPO as 4 electron-transferring phenol oxidase and oxygen. It is responsible for browning reactions throughout the phylogenetic scale.

Objective: To extract and perform enzyme assay of catalase from plant. Background information Catalase is a commonly distributed enzyme and present in approximately all organisms exposed to oxygen (such as bacteria, plants and animals). Catalysis of H2O2 into H2O and O2 is done by catalase enzyme (Goodsell, 2004).

Objective: To extract and perform enzyme assay of guaiacol peroxidase from plant. Background information: Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:

Objective: To extract and perform enzyme assay of Superoxide dismutase from plant. Background information: Superoxide dismutase (SOD, EC 1.15.1.1) is the enzyme that alternately catalyzes the dismutation (or partitioning) of the superoxide (O2-) radical into either ordinary molecular oxygen (O2) or hydrogen peroxide (H2O2). Superoxide is produced as a by-product of oxygen metabolism and, if not regulated, may cause cell damage (Hayyan, et al. 2016). Hydrogen peroxide is also responsible for cell damage and may be degraded by other enzymes like catalase. Thus, SOD is an important antioxidant defense in nearly all living cells exposed to oxygen. One exception is Lactobacillus plantarum and related lactobacilli, which use a different mechanism to prevent damage from reactive O2-.

Objective: To study peroxidase isozyme variation in rice seedlings by electrophoresis. Background information For electrophoresis and PAGE please see practical no. 4. For peroxidase please see practical no. 7.(Page 17-22) Isozymes (isoenzymes or more generally as multiple forms of enzymes) are the enzymes that are differing in the sequence of amino acids but they work on same substrate or in other words catalyze same biochemical reaction. The isozymes have difference in KM values and regulatory properties. Isozymes are very useful for proper tuning of metabolic activities and to meet the specific need of particular tissue or developmental stage, e.g. lactate dehydrogenase (LDH). Isozymes are coded by homologous genes that have diversity during evolutionary process. Although, strictly speaking, allozymes represent enzymes from different alleles of the same gene, and isozymes represent enzymes from different genes that process or catalyse the same reaction, the two words are usually used interchangeably.

Objective: To isolate DNA from plant sample by CTAB method and quantification by spectrophotometer. Background information: DNA isolation from different plant tissues viz., leaf, stem, root, flower and seed is a challenging task due to hindrance of variety of variety of biochemical compounds present and further problem increases with diverge plant species. In case of animal tissues the diversity in cellular bio-molecules in different species is much lower compared to plants. Two major phyto bio-molecules that have wide variation between species and pose serious problems in DNA isolation are: polyphenols and polysaccharides. These phyto bio-molecules if contaminated in DNA then may hinder further down applications like PCR, restriction digestion, sequencing, etc. Several methods have been developed and reported that can remove polyphenols and polysaccharides efficiently during DNA isolation from plants. For this purpose two different chemicals are widely used,the first one is CTAB (cetyl trimethyl ammonium bromide) that is a cationic detergent and helps in removal of polysaccharides and the second one is polyvinylpyrrolidone that helps in removal of polyphenols. Due to above properties the CTAB based DNA extraction buffer is very common for plant DNA extraction. Significance of different types of chemicals found in CTAB based plant DNA extraction method is explained as under:

Objective: To check quality of genomic DNA on agarose gel electrophoresis. Background information Agarose gel electrophoresis is a common method of gel based electrophoresis technique used in biochemistry, molecular biology, genetics and clinical chemistry for separation of Protein and nucleic acids from a mixture of macromolecules. Agarose is a major component of agar that is used as a matrix in gel baseelectrophoresis. The nucleic acids (DNA and RNA) are separated based on length of fragments while, proteins are separated based on size and/or charge (Kryndushkin, et al. 2003). For separation of bio-molecules the electric field is applied that leads to movement of charged particles via agarose gel matrix and bio-molecul s are separated based on difference in size in the agarose gel matrix (Sambrook and Russel, 2001). Gel casting with agarose is easy and has very few charged groups, hence found most suitable for nucleic acid separation in most of the biochemistry, molecular biology and biotechnology laboratories. Separated DNA/RNA ma be seen and recorded with stain under UV light. Further DNA fragments can be eluted from agarose gel with an ease for further down applications. The percentage of agarose gel used is in the range of 0.7 - 2. Lower percent of agarose gel is used for long DNA fragments while, higher percent of agarose gel is used for short DNA fragments with suitable running buffer.

Objective: To amplify DNA using RAPD primers and study on agarose gel. Background information: RAPD markers are decamer (10 nucleotide length) DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence and which are able to differentiate between genetically distinct individuals. RAPD is applied to analyze the genetic diversity by random primers. Unlike traditional PCR analysis, RAPD does not require any prior DNA sequence information of the target organism: the identical 10-mer primers will or will not amplify a segment of DNA, depending on positions complementary with primers' sequence. For example, no fragment is produced if primers annealed too far apart or 3' ends of the primers are not facing each other. Therefore, if a mutation has occurred in the templ that was earlier complementary with primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel. RAPD is a dominant marker means it cannot distinguish between heterozygous; single copy and homozygous; two copies. Rarely RAPD markers may be Co-dominant where different fragment length of DNA is amplified from the single locus. PCR is a molecular technique used for amplification of DNA, a complete enzyme based chemical reaction, therefore, the concentration and quality of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is notoriously laboratory dependent and needs carefully developed laboratory protocols to be reproducible. Mismatches between the primer and the template may result in the total absence of PCR product or decreased amplified product. Thus, the RAPD results can be difficult to interpret.

Objective:To transfer DNA on nylon membrane by Southern blotting. Background information A Southern blot is a method used in molecular biology to detect a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The British biologist Edwin Southern (Southern, 1975) discovered this technique the technique is known by his name. Southern blotting technique uses digested genomic DNA with restriction enzymes. Southern blotting technique may be used to identify the gene copies number in a genome. A probe that hybridizes only to a single DNA segment that is uncut with restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will likely be observed when the probe hybridizes to several highly similar sequences (e.g., may be due to sequence duplication). In the southern blotting technique a short DNA probe is used that matches based on complementary DNA sequence fragment on filter membrane. It proves that the DNA fragment on membrane contain specific sequence for which DNA probe is prepared. When DNA is transferred on the membrane from gel it permits binding of radioactive/Biotin labeled hybridization probe to the restricted DNA. Southern blotting is also important for autoradiography. Southern blotting is an important molecular biology technique that may be used for homology based cloning, screening a DNA library, screening of cloned DNA fragments and to identify methylated sites in genes.

Objective: To isolate RNA from plants using TRIzol reagent and its quantification by spectrophotometer. Background information RNA (Ribonucleic acid) is a polymeric substance present in living cells and many viruses, consisting of a long single-stranded chain of phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine, and uracil, which are bonded to the ribose sugar. RNA is necessary in all the steps of protein synthesis in all living cells and carries the genetic information for many viruses. The isolation of RNA with high quality is a crucial step required to perform various molecular biology experiment. TRIzol Reagent is a ready-to-use reagent used for RNA isolation from cells and tissues (Chomczynski, 1993).TRIzol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components. Addition of chloroform, after the centrifugation, separates the solution into aqueous and organic phases. Aqueous phase contains RNA. After transferring the aqueous phase, RNA can be recovered by precipitation with isopropyl alcohol. DNA and proteins can be recovered by sequential separation after the removal of aqueous phase. Ethanol precipitation needs DNA from the interphase, and an additional precipitation with isopropyl alcohol requires proteins from the organic phase. Total RNA extracted by TRIzol Reagent is free from the contamination of protein and DNA The isolated RNA can be further used in Northern blot analysis, in vitro translation, poly (A) selection, RNase protection assay and molecular cloning.

Objective: To check the integrity of isolated RNA on agarose gel. Background information: The most common method used to assess the integrity of total RNA is to run RNA sample on agarose gel (denaturing) stained with ethidium bromide (EtBr). While native (non-denaturing) gels can be used, the results can be difficult to interpret. The secondary structure of RNA alters its migration pattern in nativegels so that it will not migrate according to its true size. Nondenaturing conditions also result in bands that are not as sharp, and even multiple bands representing different structures of a single RNA species. Intact total RNA run on a denaturing gel will have sharp, clear 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit the 2:1 ratio of high quality RNA. Completely degraded RNA will appear as a smear (having very low mw). Inclusion of RNA size markers on the gel will determine the band size or smears size and will also serve as a good control for ensuring proper gel run. Note: Poly selected samples will not contain strong rRNA bands and will appear as a smear from approximately 6 kb to 0.5 kb (resulting from the population of mRNAs, and depending on exposure times and conditions), with the area between 1.5 and 2 kb being the most intense (this smear is sometimes apparent in total RNA samples as well).

Objective: To synthesize cDNA from isolated plant total RNA. Background information: Complementary DNA (cDNA) is double-stranded DNA made from template of single stranded RNA, [e.g., mRNA or microRNA] by reverse transcriptase enzyme. Mostly cDNA is used for cloning of eukaryotic genes in the prokaryotes. For the expression of a specific protein in a cell where the expression of that protein is not found (i.e., heterologous expression), the cDNA will be transferred that codes for the protein to the recipient cells. Retroviruses (e.g. HIV-1, HIV-2, simian immunodeficiency virus, etc.) also produce cDNA naturally and integrate in host's genome and further creates provirus (Croy, 1998). Major use of cDNA is in gene cloning or as gene probes or in the creation of a cDNA library. When scientists transfer a gene from one cell into another cell for the expression of new genetic material as a protein in the recipient cell, the cDNA will be added to the recipient (rather than the entire gene), because the DNA for an entire gene may have certain DNA that may not code for the protein or that interrupts the coding sequences of the protein (e.g., introns). Partial sequences of cDNAs are often obtained as expressed sequence tags.

Objective: To quantify aflatoxin content in given sample by Indirect competitive ELIS Background information: Enzyme linked immuno sorbent assay (ELISA) is a common biochemical technique that uses the principle of solid-phase enzyme immunoassay (EIA) for detection of substances mainly antigens in a liquid or wet sample. ELISA is a very popular technique that is used for diagnostic purpose in health sector, plant protection and also for quality test in food and other industries. This technique applies the principle of antigen-antibody reaction on a solid surface. The attached antibody is linked to certain enzymes and in the end of reaction by addition of substrate that performs enzyme-substrate reaction and makes product that has some signal mostly color change and based on intensity of color the qualitative as well as quantitative estimation of antigens can be done using ELISA reader coupled with spectrophotometer. In ELISA technique one antibody should be specific to certain antigen. For reaction of ELISA the ELISA plates are used that is also known as microtiter plate made-up of polystyrene. The sample to be tested for ELISA contain unknown amount of antigen to be detected, this sample containing antigen is immobiliz d on a solid support like polystyrene microtiter plate in two different ways; first is non-specific where antigens adsorbed to the surface and second is specific where the antigens of sample are captured by specific antibody to the antigen, like used in sandwich ELIS Once the antigens are immobilized non-specifically or specifically the detection antibody should be added that makes precise antigen-antibody complex. The detection antibody may be either covalently linked to certain enzyme or may be detected by another secondary antibody linked to bioconjugated enzyme. During ELISA steps several proteins or antibodies may non-specifically bound that can be removed by washing with mild detergent solution in between of each ELISA step. For washing of ELISA plates, ELISA plate washer may be used that mechanically wash the ELISA plates. At the end of ELISA the final wash is given and substrate is added that produces visible signal in the form of color change that indicates the presence of antigens in the sample and further quantification may be done using ELISA reader coupled with spectrophotometer.

1.Which one is the example of abiotic stress? a.Drought b.Salinity c.Flood d.All of these 2.Which one is the example of osmoticum? a.NaCl b.PEG c.H2O