eChapters

Indian Agriculture is facing a wide spectrum of constraints such as burgeoning population, shrinking farm land, restricted water availability, imbalanced fertilization, low soil organic carbon, besides experiencing the fatigue of green revolution and vagaries of climate change. To address all the challenges ahead, we should think of an alternate technology such as “nanotechnology” to precisely detect and deliver the correct quantity of nutrients or other inputs required by crops that promote productivity while ensuring environmental safety. The word “nano” refers to the size of one-billionth of a metre or one-millionth of a millimeter in any one of the dimension. We are aware that all materials are made up of atoms which are the smallest units and for example, ten hydrogen atoms put together will measure one nanometer. As the size gets reduced, the surface area gets increased by several folds.

Introduction

DNA sequencing is the process of determining the nucleotide order of a given DNA fragment. The most commonly used sequencing method is the dideoxy method. This method uses dideoxynucleotide triphosphates (ddNTPs) which have an H on the 3’ carbon of the ribose sugar instead of the normal OH found in deoxynucleotide triphosphates (dNTPs). Dideoxynucleotides are chain terminators. In a synthesis reaction, if a dideoxynucleotide is added instead of the normal deoxynucleotide, the synthesis stops at that point because the 3’OH necessary for the addition of the next nucleotide is absent (Sanger et al., 1977).

Introduction

Only after the discovery of restriction endonucleases it became possible to digest any DNA molecule at a specific site and any two pieces of DNA could be joined together with the help of another modifying enzyme, the DNA ligase. Consequently, a new branch of molecular biology, namely genetic engineering has been developed. With the help of genetic engineering techniques, it is now possible to identify and isolate the gene (for a protein of interest and insert it into the genome of another organism with the help of a suitable vehicle. The foreign gene thus cloned, can be amplified to produce millions of its copies. The insert gene can be expressed by the host to synthesize the gene product. The technique can also be used for understanding the regulation of expression of the cloned gene. The possibilities are endless and probably sky is the only limit. In this chapter we will briefly discuss the basic principles and protocol involved in gene cloning.

Southern Blotting
Introduction

Southern hybridization was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. It is also called Southern blotting since the procedure for transfer of DNA from the gel to the nitrocellulose filter resembles blotting. In Southern hybridization, a sample of DNA containing fragments of different sizes is subjected to electrophoresis using either polyacrylamide or agarose gel.

Introduction

Restriction enzymes, recognizes specific base sequences in double-helical DNA and cleaves at specific places, both strands of a duplex containing the recognized sequences. To biochemists, these exquisitely precise scalpels are marvelous gifts of nature. They are indispensable for analyzing chromosome structure, sequencing very long DNA molecules, isolating genes, and creating new DNA molecules that can be cloned. Werner Arber and Hamilton Smith discovered restriction enzymes, and Daniel Nathans pioneered their use in the late 1960s.

Introduction

Gel electrophoresis is a technique for separating charged molecules with different sizes. Two kinds of gels are commonly used: agarose and polyacrylamide. Agarose gels can be applied to a wider range of sizes than polyacrylamide gels.  By using standard agarose electrophoresis, nuclei acids up to 50 kb may be separated. If Pulsed Field Gel Electrophoresis is used, the upper limit can be extended to 10 Mb. Polyacrylamide gels may separate nucleic acids that differ in length by only 1 nucleotide if their length is less than 500 bp (Sambrook et al., 1989).

Introduction

This chapter gives first hand information on PCR for beginners. PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single gene, or just a part of a gene. As opposed to living organisms, the PCR process can copy only short DNA fragments; usually up to 10 kb (kb stands for kilo base pairs). Certain methods can copy fragments up to 40 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell-for example, a human cell contains about three billion base pairs (Kary Mullis, 1983).

Introduction

In order to use a fine quality of DNA for various molecular biological work like sequencing, cDNA synthesis and cloning, RNA transcription, transfection, nucleic acid labeling (random primer labeling), etc., quality and quantity of DNA is extremely important to have the defined template concentration. Failure to produce results from the above techniques can sometimes be attributed to an incorrect estimate of the DNA template used.

DNA Isolation Protocol
Introduction

Most of the all procedures involved in molecular biology experiments require the purified form of nucleic acid. It forms a fundamental requirements for genome characterization, gene mapping and for identification and isolation of gene for genetic engineering. The most important step involved in removal of proteins which is carried out in different ways. The following protocol is one of the most commonly used protocols in animal, plant and microbial samples.

Plant cell culture
Introduction

As the human population is expanding there has been increasing concern over whether current rates of food productions can support the growing population. In response to the increasing demand, scientists and farmers have been working hard to come up with new ideas to meet these needs. These ideas range from simple procedures including selection of the best seeds to more complex procedures requiring the transplantation of genes into different plant species. This chapter focuses on one of the important aspects of plant science which is the growth of plant cells in an artificial media. This process is known as plant cell culture and can be applied in many different ways. 

Introduction

This section of the manual contains instructions for preparing stock solutions that are commonly used in laboratory protocols for the analysis of DNA, RNA and proteins and visualization of gene or protein expression. Each of these stock solutions is provided as either the most convenient concentration or the most frequently used stock concentrations to be used in the protocols of an individual laboratory.

All chemicals must be reagent grade or molecular biology grade, and the water used in the preparation of all solutions must be the highest quality available. Use sterile-glass distilled deionised water whenever possible. Unless otherwise stated, most solutions required sterilization either by filtration or autoclaving.

Molecular biology labo-ratories are becoming very common in every University and research institutes and a good laboratory manners is extremely important as many of the equipments are very expensive and sensitive. Hence familiarity with equipments becomes very crucial before starting any experiment.

Summary

Lac is a resinous exudation from the body of female scale insect Laccifer lacca Kerr. belongs to family Lacciferidae of phylum Arthropoda, class  Insecta and order Hemiptera. The lac insect lives on native trees in India, China,Thiland, Burma/Myanmar and Malaysia. In India it is chiefly grown on trees like Kusum, “Palas”, and “Ber. It is also purposely cultured on shoots of these tree species. Lac cultivation is an alternative agricultural profession and provides subsidiary source of income for a large number of farmers mainly in Jharkhand, Chhattisgarh, West Bengal, Odisha, North-East states and other parts of the country. India, which is the highest producer of lac, contributes around 55% of the total world requirement followed by China and Thiland. The country exports around 80-90% of its production.

Summary

Nanotechnology is an interdisciplinary areas of science and technology which refers broadly to a field of applied science and technology whose unifying theme of control of matter is on the molecular scale that integrates material science and biology. Due to their advantages over non-biological systems, several research groups have explored the use of biological systems for the synthesis of nanoparticles. Among the different microbes used for the synthesis of nanoparticles, fungi are efficient candidates for fabrication of metal nanoparticles both intra-and extracellulary. The nanoparticles synthesized using fungi present good polydispersity, dimensions and stability. The potential applications of nanotechnology and nanoparticles in different fields have revolutionized the health care, textile and agricultural industries and  they are described in present chapter.

Summary

Molecular evolution is the process of evolution at the scale of DNA, RNA, and Protein. Molecular evolution emerged as a scientific field in the 1960s as researchers from molecular biology, evolutionary biology, and population genetics sought to understand recent discoveries on the structure and function of nucleic acids and protein. It covers two broad areas of study : the evolution of macromolecules, and reconstruction of the evolutionary history of genes and organisms. The evolution of macromolecules refer to the characterization of the changes in the genetic material (DNA or RNA sequences) and its products (Proteins of RNA molecules) during evolutionary process, and to the rates and patterns with which such changes occur, and also the mechanism responsible for such changes. The second area also known as “molecular phylogenetics”, deals with the evolutionary history of organisms and macromolecules as inferred from molecular data and the methodology of tree reconstruction.

Summary

One of the most seductive lures of the genomic revolution is the promise of personalized medicine. The rapid identification of tens of thousands of human genes and hundreds of thousands of DNA variations that might influence disease susceptibility has spawned a new field Pharmacogenetics and Pharmacogenomics deal with the genetic basis underlying variable drug response in individual patients. The traditional pharmacogenetic approach relies on studying sequence variations in candidate genes suspected of affecting drug response i.e. single or few genes. On the other hand, pharmacogenomic studies encompass the sum of all genes, i.e., the genome. Numerous genes may play a role in drug response and toxicity, introducing a daunting level of complexity into the search for candidate genes. The high speed and specificity associated with newly emerging genomic technologies enable the search for relevant genes and their variants to include the entire genome.

Summary

Substance that acts as a catalyst in living organisms, regulating the rate at which life’s chemical reactions proceed without being altered in the process. Enzymes reduce the activation energy needed to start these reactions, without them, most such reactions would not take place at a useful rate. Because enzymes are not consumed, only tiny amounts of them are needed. Enzymes catalyze all aspects of cell metabolism, including the digestion of food, in which large nutrient molecules (including proteins, carbohydrates, and fats) are broken down into smaller molecules; the conservation and transformation of chemical energy and the construction of cellular materials and components. Almost all enzymes are proteins; many depend on a nonprotein cofactor, either a loosely associated organic compound (e.g., a vitamin) or a tightly bound metal ion (e.g., iron, zinc) or organic (often metal-containing) group.

Summary

The process of administration of a drug into the human body to cure a disease is regarded as drug delivery. The common routes of administration of the drugs include oral, nasal, inhalational, transdermal, dermal, ocular, rectal and vaginal. In the current chapter, an attempt has been made to discuss about the various methodologies which are being used to deliver the drug to the human patients.

Summary

Human being is dependent on nature and natural resources to combat diseases from time immemorial. Ayurveda describes the use of traditional herbs in medicinal preparations in ancient India. Such types of evidences are there in classical Chinese and Greek records also. Nowadays, modern tools are used to identify and purify the active principle from the herbal composition having the therapeutic effect. Bioinformatics tools are often used to screen the drug efficacy of natural products. Using computational and Bioinformatics tools, the efficacy of lead molecules can be screened. Moreover, drug targets can be identified using Bioinformatics and proteomics approach. QSAR and molecular docking tools are used to study the probable activity and mode of action of drug molecule. This approach has minimized the cost and time involved in the drug discovery process.

Summary

The present era is witness of understanding the science of human diseases at molecular level. The advances of science in terms of Genomics, Proteomics, Metaboliomics, Lipidomics and bioinformatics have drawn an excellent pathway to discover more promising drugs. It’s still a challenging task to reduce the time line to discover new drug, which is approximately 10-15 years. A drug discovery program requires billions of dollars but without the promise of success. After screening of thousands of compounds only one becomes successful in clinical trials and reaches to the market. Although there are lot many ways to approach drug discovery, nature has gifted many interesting compounds to fight the diseases. On the other hand isolation of pure compounds from natural products like bacteria found in soil or different parts of plants is itself a challenging area. The modern fashion of natural product isolation is guided by bioactivity. Hence a feasible assay to access the bioactivity has got a great importance.