Protocols used in Molecular Biology

Genomic DNA extraction is the starting point for various downstream molecular biology applications viz. PCR, restriction analysis, hybridisation etc. Numerous problems like DNA degradation, co-isolation of viscous polysaccharides, polyphenols and other secondary metabolites causing damage to DNA, inhibiting restriction enzymes, DNA polymerases etc, are routinely encountered during DNA isolation from plants. Quinone compounds resulting from oxidation of polyphenols lead brown the DNA preparations and can also damage proteins and DNA`s due to their oxidizing properties. This results in a poor yield of high molecular weight DNA. The protocol below explains the extraction of DNA via the CTAB method, involving three major steps viz lysis of cell wall and membranes, extraction of genomic DNA and precipitation of DNA.

The preparation of intact ribonucleic acid is difficult because of the action of nucleases, which are liberated upon tissue homogenisation. In many cells, high concentrations of the ribonucleases are reserved in the secretory granules and upon disruption of the cell, they get mixed with the RNA and lead to its degradation. Guanidinium chloride and thiocyanate are potent chaotropic agents that reduce hydrophobic interactions and disrupt protein tertiary structures, disassociate proteinnucleic acid complexes and disintegrate cellular structures. Guanidinium thiocyanate is especially strong protein denaturant because both the cation and anion disrupt the hydrophobic bonds between the amino acid side chains. RNA usually binds to proteins within a cell and this agent disassociates the nucleoprotein complex, without disrupting RNA structure. Thus RNA can be obtained by using these agents, after homogenisation and low-speed centrifugation and precipitated with ethanol. The protocol below explains the stepwise isolation of total RNA from cells and tissues using TRIzol reagent which is the mono-phasic solution of phenol and guanidine thiocyanate.

Real-time PCR offers a wide area of application to analyze the role of gene activity in various biological aspects at the molecular level with higher specificity, sensitivity and the potential to troubleshoot with post-PCR processing and difficulties. With the recent advancement in the development of functional tissue graft for the regeneration of damaged/diseased tissue, it is effective to analyze the cell behaviour and differentiation over tissue construct toward specific lineage through analyzing the expression of an array of specific genes. With the ability to collect data in the exponential phase, the application of Real-Time PCR has been expanded into various fields such as tissue engineering ranging from absolute quantification of gene expression to determine neo-tissue regeneration and its maturation. In addition to its usage as a research tool, numerous advancements in molecular diagnostics have been achieved, including microbial quantification, determination of gene dose and cancer research. Also, in order to consistently quantify mRNA levels, Northern blotting and in situ hybridization (ISH) methods are less preferred due to low sensitivity, poor precision in detecting gene expression at a low level. An amplification step is thus frequently required to quantify mRNA amounts from engineered tissues of limited size. When analyzing tissue-engineered constructs or studying biomaterials–cells interactions, it is pertinent to quantify the performance of such constructs in terms of extracellular matrix formation while in vitro and in vivo examination, provide clues regarding the performance of various tissue constructs at the molecular level. In this chapter, our focus is on Basics of qPCR, an overview of technical aspects of Real-time PCR; recent Protocol used in the lab, primer designing, detection methods and troubleshooting of the experimental problems.

Western blots (WB) are designed to investigate protein levels and their patterns of modification in homogenized tissue samples. Although, Western blots are quantifiable, unlike immunohistochemistry, cellular integrity is lost. The availability of antibodies against the protein and their patterns of modification of interest form the basis of both Western blots and Immunohistochemistry. Antibodies can also be directed not only against proteins but against chemical modifications of the proteins too, such as phosphorylation and glycosylation of specific amino acid residues. In Western blotting, the proteins in the sample are denatured, size-separated on a denaturing acrylamide gel, and transferred to a nylon membrane. Antibody paratopes can then bind to the antigenic epitope in the protein present on the nylon membrane. Thus, with the help of a chemiluminescent assay system that darkens X-ray films, the resulting antibody-antigen complex can be visualized. Because of the ubiquitous and relatively inexpensive availability of WB equipment, the quality of WB in publications and following analysis and investigation of the data can be variable, possibly resulting in forged conclusions. This may be because of the poor laboratory technique and/or lack of understanding of the significant steps involved in WB and what quality control procedures should be followed to ensure effective data generation. The present book chapter focuses on providing a detailed description and critique of WB procedures and technicalities, from sample collection through preparation, blotting, and detection, to examination of the data collected. We aim to provide the reader with the improved expertise to decisively carry out, assess, and troubleshoot the WB process, in order to produce reproducible and reliable blots.

Immunohistochemistry (IHC) is a well-known technique in the field of biological and medical sciences. This technique is based on the principle of antigenantibody interaction and is used for identification of cellular or tissue constituents, i.e., an antigen by using a specific antibody. The binding of an antibody to an antigen is confirmed either by labelled primary antibody itself or by using secondary labelling method such as fluorescence labelled antibody. Such interactions give information about the cellular process occurring inside the cell. In last few years, huge amount of data have been generated using IHC. Furthermore, adequate knowledge of this technique is required for the optimum result and its reproducibility. The detailed information about the tissue section, antigen retrieval (AR), increased sensitivity of the detection systems and proper standardization are the key points for this technique. This protocol will address overview of the technique, tissue preparation, microtome, antigen retrieval, antibodies and antigen fixation, detection methods, background reduction and trouble shootings.

Soybean (Glycine max) is one of the legumes, susceptible to yellow mosaic disease caused by Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) infection. The quantitative proteomic analysis allows achieving deeper knowledge about the viral infection. For quantitative proteomic analysis, two-dimensional gel electrophoresis (2D-PAGE) is the common method of choice. Optimization is required even for the published protocols based on the type of sample to be analyzed and for the proteins of interest. We compared four different published protocols with some modifications and selected the one which is more effective in terms of resolution and reproducibility of 2D-PAGE. Here we present our simple and cost-effective procedure for the detection of viral infection and proteomic analysis of YMV infected soybean leaves without compromising the resolution and reproducibility of 2D-PAGE.

In the recent past, two dimensional gel electrophoresis has emerged as a powerful molecular biology tool for the comparative expression profiling of complex protein sample. It involves the separation as well as the resolution of diverse proteins sample on the basis of isoelectric points and molecular mass of protein in two dimension ways. In this way, it reflects the view of overall proteome status including differentiation in protein expression levels, post-translational modifications etc. Moreover, this allows the identification of novel biological signatures, which may give a particular identity of pathological background to cells or tissues associated with various types of cancers and neurological disorders. Therefore, by utilizing such tools, one can clearly investigate and compare the effects of particular drugs on cells of tissues and also one can analyze the effects of disease on the basis of variations in protein expression profile at broad spectrum. Recently, to get more error-less and accurate proteome profile, conventional 2-D gel electrophoresis has been enhanced with the inclusion of different types of protein labeling dyes which enables a more comparative analysis of diverse protein sample in a single 2-D gel. In this advanced technique (2-D-DIGE), protein samples are labeled with three different types of CyDyes (Cy2, Cy3, and Cy5) separately and combined and further resolved on the same gel. This will facilitate the more accurate spot matching on a single gel platform and will also minimize the experimental variations as commonly reported in the conventional 2D-gel electrophoresis. Therefore, in the present proteomic research era, 2D-DIGE has proved to be an extremely powerful tool with great sensitivity for up to 125 ng of proteins in clinical research volubility especially, neurological and cancer related disorders.

Genotyping is a process of determining the genetic constituent/genetic makeup “genotype” of an organism by examining the individual DNA sequence and comparing to a reference or other individual sequence. It helps the researchers to explore the genetic constitution, genetic linkages or variations like Single Nucleotide Polymorphisms (SNP) or multi-nucleotide changes in DNA. Identification of genotypes is also useful for determining their role in phenotypic expressions. Genotyping is an essential tool for researchers to find out disease-associated genes and gene variants. Genotype determined can also be used for the identification of susceptibility and prognosis for any disease and to find out responders/non-responders for a specific treatment, thus leading the way towards personalized medicine. Several molecular techniques have provided swift, reliable and accurate ways for determining genotypes. The process of genotyping involves molecular techniques like isolation and quantification of genomic DNA, visualization of DNA on agarose/polyacrylamide gel using electrophoresis, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), random amplified polymorphic detection (RAPD) of genomic DNA, amplified fragment length polymorphism (AFLP), sequencing, allele-specific oligonucleotide (ASO) probes, microarrays etc. The present chapter will describe the protocols for different molecular techniques that are used to determine genotypes.

The regulation of transcription and translation of a gene under a given environment is dependent on several factors and epigenetics is one such factor, responsible for the differential expression of several genes in health and in various diseases. DNA methylation, an important epigenetics mechanism has been shown to play a vital role in numerous cellular processes, and the abnormal patterns of methylation have been linked to the number of human diseases. CpG islands, a short stretch of DNA enriched with CpG sites in the 5’ end of a gene, although remains unmethylated but tends to methylate aberrantly upon certain environmental exposures. The methylation of the promoter region bearing transcriptional start sites of those genes that encodes tumor suppressors such as tumor protein p53, retinoblastoma-associated protein 1, tumor protein p16, breast cancer 1 and many more result in the reduced expression of these genes and have been implicated in a large number of cancers like retinoblastoma, colon, lung and ovarian. A growing number of human diseases have been found to be associated with the aberrant DNA methylation. Hence, a deep insight into the individual’s epigenetic profile is the need of the hour. Several approaches have been developed to map DNA methylation patterns genome-wide. Some of these approaches include enzymatic digestion with methylation-sensitive restriction enzymes, the capture of 5-mC by methylated DNA-binding proteins followed by nextgeneration sequencing and methyl-DNA immunoprecipitation followed by sequencing of precipitated fragments. However, this chapter is going to describe the most recommended method for studying DNA methylation pattern, the method based on bisulfite sequencing. The bisulfite treatment of DNA converts unmethylated cytosine(s) to uracil(s), which are subsequently amplified as Ts by PCR. Hence, the bisulfitetreated DNA has mutations specifically at unmethylated Cs that can be mapped by Next-Generation sequencing.

Chromatin immunoprecipitation or ChIP is an excellent method of investigation of the specific protein interaction and its altered forms with DNA region. These interactions have a significant role in various cellular processes such as replication, transcription, DNA damage repair, genome stability, gene regulation and segregation at mitosis. This technique is therefore giving us power to study a variety of cellular mechanisms inside the cell in terms of protein-DNA interaction. As the name Chromatin immunoprecipitation suggests this method utilizes chromatin preparation from cells to selectively immune-precipitate the protein of interest to identify DNA sequence associated with it. Chromatin is an organized structure of eukaryotic DNA which contains double-stranded DNA wrapped around nucleosomes. ChIP has been extensively used to depict transcription factors, variants of histone, chromatin modifying enzymes, post-translational modification of histone on the genome. In the classical ChIP method, protein and DNA is irreversibly cross-linked by UV exposure followed by immunoprecipitation with specific antibodies, protein-DNA complex is then purified, treated with proteases and then analysis is done by the method of Southern blot or dot blot using a radio-labelled probe derived from the cloned DNA fragment of interest. Further, it was modified by using formaldehyde for reversible cross-linking of protein-DNA complex and polymerase chain reaction for the detection of fragments of precipitated DNA. ChIP is a cumbersome procedure to perform and present many limitations, for example it requires many cells. Therefore, many modifications and variations, have also developed with the time which enables us to simplify the procedure and widen its range of applications. This chapter provides a brief method for Chromatin immunoprecipitation (ChIP) and its applications.

Osteosarcoma is the most common type of malignancy of bone and generally occurs among adolescent and young adults. Like the osteoblast cells of normal bone, osteosarcoma also forms the bone matrix, but the osteoid is not as strong as that of normal bones. Osteosarcoma is characterized by the production of weak or immature bones by the malignant cells. As the diagnosis of osteosarcoma is extremely poor, it suggests a critical need to develop some promising anti-osteosarcoma drugs to improve disease outcome. Many anti-cancer compounds induce apoptotic cell suicide via some potent cellular, molecular and biochemical markers. The cancer cell lines provide a valuable model system to study an extensive variety of cancer characteristics in the cell biology, genetics and chemotherapy or the impact of therapeutic molecules. The methods presented in this chapter describe the experimental technique used to culture the osteosarcoma cells for the documentation of DNA fragmentation and Caspase-3 activation associated with apoptosis.

Ovarian carcinoma is the 5th most common type of cancer of gynecologic origin and accounts for about one-fourth of the total malignancies of the female genital tract. Ovarian carcinoma accounts for highest mortality in females due to the development of chemo-resistance against drugs and lack of symptoms and undetectable biomarkers in the early stages of diagnosis. Tumour debulking, chemotherapies, radiotherapies, targeted therapies, immunotherapies and stem cell transplants are some of the measures that have been adopted by the experts for curing the disease but still, full control over the problem has not been achieved. Research on various herbal and chemosynthetic nano-compounds have shown a new light in this regard, as the studies on them so far have revealed that they have anti-proliferative and apoptotic properties that will help in finding new ways to develop drugs for cancer patients. This chapter deals how to culture and maintain the human ovarian carcinoma cell lines in the laboratory which are being procured from cell repositories and then to study the anticancer efficacy of various promising compounds by potent molecular markers like cellcycle progression and annexin V- FITC apoptosis detection.

The advent of advanced microscopes; during microscope evolution from simple microscopes to confocal and live cell microscope; having digital imaging facility revolutionized our view for the living cells. In the protein localization study, fluorescent proteins are tagged at amino or carboxyl (preferably) terminal of desired protein for live cell study. These live cell studies improved our understanding of protein dynamics and understanding its role in biological regulation. The mutational variants of fluorescent tags (GFP, RFP); can be used with different protein; which will efficiently use UV-Visible to Far Red light spectrum; without overlapping of excitation and emission spectrum. Further, various cell organelle (Lysosome, Golgi bodies, Endoplasmic Reticulum, Mitochondria, Nucleus) trackers; improved our live cell localization studies in the wide non-overlapping UV-Visible spectrum.This chapter gives an overview for live cell protein localization study in mitotically active, unicellular stage of Dictyostelium discoideum. This evolutionary cutting edge organism had both unicellular as well as multicellular stages during its life cycle. This chapter will provide the design of fusion of fluorescent tag to the specific gene and its live cell localization. Further, it will cover; transformation of the unicellular organism; drug based selection; sample preparation with nuclear, mitochondrial localization markers (trackers) and live cell localization study on live cell-confocal microscope setup. It will also have a glimpse of the design of fusion protein with an aspect of advantage and disadvantages.

The foundation knowledge of recent advancements of neuroscience was based on the Golgi staining observations. This is one of the best approaches to visualise the neuronal cytoarchitecture and complete morphology of neurons with incomparable clarity. This technique is based on the principle of heavy metal impregnation. There are many modifications and advancement occurred to improve the visualization. This chapter will provide the recently used protocols to visuals the neuronal architecture, dendritic arborization and spine density in different brain regions. Along with the manual observation, the present chapter also describes the currently used tools and software for the better understanding and visualisation of neurons.

Medicinal plants are major sources of secondary metabolites for which they have been paid more attention by pharmaceutical industries. In order to produce these secondary metabolites, medicinal plants are cultivated and for that plant tissue or organ, culture can be a suitable alternative. However, these plants are treated with plant hormones and elicitors to enhance the secondary metabolites and such elicitation may lead to genetic or epigenetic changes which are known as somaclonal variations. Thus, a stringent method of monitoring is required to observe the true-to-types of these medicinal plants when multiplied through tissue culture. Molecular markers like Randomly Amplified Polymorphic DNA (RAPD), Inter-Simple Sequence Repeat (ISSR), and Simple Sequence Repeats (SSR) are highly suitable markers to assess clonal fidelity in micropropagated medicinal plants. In the present chapter, the execution of such markers to check somaclonal variations in tissue culture raised medicinal plants is discussed in detail.