eChapters

DNA Sequencing

DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA. Sequencing can be done on a short fragment of DNA to cover the entire genome. The maps thus constructed using the nucleotide sequences in the correct order is called as sequence map.

In 1977, twenty-four years after the discovery of the structure of DNA, two separate methods for sequencing DNA were developed: the chain termination method (Sanger et al., 1977) and the chemical degradation method (Maxam and Gilbert, 1977). Both methods were equally popular to begin with, but, for many reasons, the chain termination method is the method more commonly used today. In chemical degradation method, chemicals specific to each base remove the last base, which has been labelled. The method uses toxic chemicals which required high care while handling.

In chain termination method, DNA polymerase extends a DNA strand in which dideoxynucleotide becomes incorporated terminating the chain extension (phosphodiester formation) at the 3’OH site making an array of fragments differing in length. This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis.

DNA can be sequenced in both double stranded and single stranded templates by cloning in plasmid or phage vectors. There are two sequencing methods.

Principle

Determination of the precise sequence of nucleotides in a sample of DNA is called “DNA Sequencing”. The most popular method for doing this is called the “Dideoxy method or Sanger Method” (named after Frederick Sanger, who was awarded the 1980 Nobel Prize in chemistry for inventing it). DNA is synthesized from four deoxynucleotide triphosphates. The top most figure is a normal nucleotide: deoxythymidine triphosphate (dTTP). During polymerization each new nucleotide is added to the 3' -OH group of the last nucleotide added.

The dideoxy method has been coined after the synthetic nucleotides that lack the –OH at the 3’carbon atom of the sugar and it plays an important role in this process atom (red arrow). In the figure below dideoxythymidine triphosphate — ddTTP, a dideoxynucleotide has been shown that can be added to the growing DNA strand. But the chain elongation ceases when the next nucleotide tries to get attached to the former as there is no 3' -OH for the next nucleotide to be attached to. Hence, the dideoxy method is also called the chain termination method. 

Identification of DNA as the Genetic Material

Experiments with pneumococcus suggested that DNA is the genetic material

Hershey and Chase provided evidence that the genetic material injected into the bacterial cytoplasm is T2 phage DNA

RNA functions as the genetic material in some viruses Work Before Watson and Crick

Nucleotides are the building blocks of nucleic acids

Nucleotides are linked together to form a strand

In 1869, Miescher first isolated DNA and named it “nuclein.”

In 1902, Archibald Garrod linked heredity to enzyme (protein) defects. In 1909 he published a book entitled “Inborn Errors of Metabolism.”

In 1928, Griffith identified a “transforming factor” that transmitted infectiousness in bacteria.

In 1944, Avery, MacLeod and McCarty showed that the transforming factor was DNA)

In 1950, Hershey and Chase, using their famous “blender” experiment, confirmed that DNA, not protein, is the genetic material.

The work of chemist Phoebus Levene showed that DNA includes deoxyribose, nitrogenous bases, and phosphates, in equal proportions.

Chargaff s data showed that the number of purines equals the number of pyrimidines.

Using Rosalind Franklin’s X-ray diffraction patterns, Watson and Crick deduced that DNA is a double helix

In 1868, Johann Friedrich Miescher, a young Swish medical student got excited with an acidic substance that he isolated from pus cells obtained from bandages used to dress human wounds. To obtain large quantities of pus for his experiment, Miescher began purchasing used bandages from surgical clinics in Tuebingen, Germany. He separated pus cells from bandages and treated the cells with pepsin, a proteolytic enzyme that he isolated from stomach of pigs. After pepsin treatment, he recovered an acidic substance that was named as “nuclein” by him. This nuclein was unusual in the sense that it was rich in nitrogen and phosphorous, two elements known at that time to coexist only in certain types of fat. Miescher’s paper on this investigation was published in 1871. At that time, importance of Miescher’s nuclein was not fully appreciated. Even the existence of polynucleotide chains, the key component of the acidic material in Miescher’s nuclein was not documented until 1940s. The role of nucleic acid in storing and transmitting genetic information remained un-established until 1944 and the double-helix structure of DNA was not discovered until 1953 by James Watson and Francis Crick. Even in 1953, many geneticists remained skeptical to accept the role of nucleic acid as the genetic information carrier rather than proteins as nucleic acids exhibited less structural variability than the proteins (Snustad et al., 1997).

A laboratory is essential for research work in science and development. Most of the research institutes have dedicated laboratories for researchers and students to conduct experiments, perform research work, as well as to provide hands-on learning/training to students. There are certain rules and regulations laid down by the laboratory in-charge which need to be followed strictly for safety purposes. Some of the laboratories keep ‘safety manual’ for researchers to read before entering into the laboratory to conduct experiments. Whenever safety is compromised in a laboratory, there is increase in risk of unwanted incidents in the laboratory. Storing food/beverages in the same refrigerator where laboratory chemicals are kept may lead to disastrous effects on health. This chapter aims to provide instructions in brief on the things to be performed (Do’s) before and after entering onto a laboratory as well as the things never to be done (Don’ts) in a laboratory for the safety reasons.

Good Laboratory Practices (Do’s)

• P Wear apron/labcoat while working in the laboratory.
• P Use gloves/respiratory mask/fumehood while handling toxic/volatile/inflammable chemicals/reagents.
• P Use pippet or pippetor for safety in transfer of solutions.
• P Protect eyes, face and naked parts of the body from UV radiations. Use safety goggles or face mask/apron.
• P Keep your eyes away from Liquid Nitrogen and use cryogloves while handling it.
• P Arrange all the necessary materials/solutions before starting the experiment.
• P Periodically fumigate/ozonize culture and inoculation rooms.
• P Autoclave all contaminated materials before discarding.
• P Pre-treat gel or solution containing Ethidium Bromide (EtBr) before discarding into laboratory waste-disposal bin, and use gloves while handling them.
• P Burn all the transgenic plant materials in an incinerator after taking necessary observations.
• P Make entry in the designated log-book and clean the instrument after using it properly.
• P Switch-off the instrument after use or when not in use.
• P Wash hands with soap and remove the labcoat before leaving/going out of the laboratory7.
• P Ensure that switches for fans/lights, and water taps are off while leaving the laboratory.
• P Follow the biosafety guidelines suggested for handling and disposal of the hazardous chemical.

Introduction
Domestication is the process of adapting wild plant species for human cultivation and consumption and it has played a pivotal role in shaping modern agriculture. Since the dawn of civilization, humans have selectively bred plants to enhance desirable traits, such as increased yield, improved taste, and resistance to pests and environmental stresses. Through domestication, early agriculturalists inadvertently altered the genetic makeup of wild progenitors, favouring traits that aligned with their needs and preferences. This process involved selecting and propagating plants with desired characteristics over successive generations gradually transforming once-unruly wild species into more docile, productive and palatable varieties. The domestication of crop plants was a gradual and ongoing process, often spanning thousands of years. It required keen observation experimentation, and an inherent understanding of plant biology and genetics even before these concepts were formally understood. This patient and persistent effort by our ancestors paved the way for the development of diverse and nutritious food sources, enabling the establishment of sedentary communities and the rise of complex civilizations. While modern breeding techniques have advanced significantly, the fundamental principles of domestication remain a cornerstone of crop improvement efforts. By understanding the historical significance and underlying mechanisms of domestication, we can better appreciate the invaluable contributions of our ancestors and continue to build upon their legacy, ensuring a sustainable and diverse food supply for generations to come.
The current state of food systems is being challenged due to the heavy reliance on a limited number of resource-intensive staple crops. The emphasis on maximizing yield and the consequent reduction in diversity over the course of domestication history has led to the development of contemporary crops and agricultural practices that are environmentally unsustainable, susceptible to the impacts of climate change, lacking in essential nutrients, and contributing to social inequalities. For many years, researchers have advocated for diversification as a viable solution to tackle the issues surrounding global food security. In this context, we present a vision for a new phase of crop domestication, which aims to expand the range of crop diversity. This approach involves active engagement and benefits for three key components of domestication: a) the cultivated crops themselves, b) the surrounding ecosystems, and c) the human populations depending on these crops. We delve into the potential of current tools and technologies to initiate a revival of diversity within existing crops, enhance the value of underutilized crop varieties, and introduce new crops into cultivation. This multipronged strategy aims to reinforce genetic diversity, promote resilient agroecosystems, and establish a more varied and robust food system. The successful execution of this renewed phase of domestication hinges on a bold commitment from researchers, funders, and policymakers to invest in both fundamental and practical research efforts. As we navigate the challenges of the anthropocene epoch, characterized by significant human impact on the environment, the process of domestication offers a pathway to construct more diverse and adaptable food systems that can better serve humanity’s needs.

The main purpose of this lecture note is to provide basic information on patent drafting skills and train applicants for drafting a patent in clear and concise manner.

We will see the components of Complete Specification through the following Case studey (1) of a solar water heater. The figures are provided below for reference and better understanding.

Introduction

Drought is the most common adverse environmental factor which limits crop production in different parts of the world. Often drought is accompanied by relatively high temperatures, which promote evapotranspiration, and hence could accentuate the effects of drought and thereby further reduce crop yields. Since these events occur more frequently in tropical and semitropical regions where most of the developing countries are situated, droughts are often associated with food shortages and an overall setback to developmental activities. Therefore, raising of drought-resistant crops is common sense to achieve stability in production and to enhance the possibility of self-suficiency in food. Consequently, breeding for drought resistance is a major objective of many research programmes in the international and national institutions. This has been so for the past several decades. But, it is disappointing that, there has not been any measurable success in these programmes compared with the success achieved in breeding for yield; or breeding for disease resistance or breeding for quality attributes. In fact, this was amply expressed by Arnon (1980) when he stated “Breeding for drought resistance has been a consistent theme for as long as I remember and probably the greatest source of wasted breeding efforts in the whole field of plant breeding.” However, the fact remains that we do hear of drought resistant varieties in many crops. For example, C-306 in wheat, Lalnakanda in rice and M 35-1 in Sorghum are some of the better known examples in India. It is also claimed that a large number of cultures among the Assam Rice Collection exhibit a considerably high degree of drought tolerance. Many land races and wild relatives of several crop species are said to contain drought resistant traits, which could be profitably utilised in breeding programmes. This situation is not unique to India alone but is a common experience in different parts of the world. However, to afirm a conviction a short questionnaire to assess the current state-of-art on breeding for drought resistance was distributed to 75 distinguished plant breeders and plant physiologists all over the world. The questionnaire and a summary based on the responses is as follows:

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.

Abstract

Drug design is a complex computational process that involves studying the structural and physicochemical properties of biological moieties in order to identify new chemical or biochemical substances that can be used as drugs with specific pharmacological properties. The drug discovery pipeline is a crucial aspect of drug design that requires a detailed understanding of the various phases involved. In this chapter, we discuss the fundamental principles and phases of drug design. Additionally, we delve into the concept of docking, a critical step in the drug discovery process that involves predicting the orientation and affinity of a ligand binding to a protein target. The use of computational tools and techniques in drug design has revolutionized the field and is essential for the development of safe and effective drugs.

Introduction

While experimenting with chemicals, sometimes we need to dry the chemical/ solution (solid, liquid or gas) using a process like evaporation or distillation, mainly to remove water or different solvents. Drying is the process of evaporation or distillation to remove water/solvent from a solution, solid or even from gas. Generally, a drying agent (usually an anhydrous inorganic salt) is used for this process, which reacts with the water (present in the chemical/ solvent to be dried) to form a hydrate. For example, anhydrous MgSO4 is one of the drying agents. The drying agents are recognized by their capacity (the rate at which they absorb water) and their intensity (the amount of water left behind in the solvent at equilibrium). No chemical reaction or change in phase occurs during the drying process.

Type of Solvents

Apart from water (a universal solvent), there are three families of organic solvents used in laboratories.

1) Oxygenated solvents: Alcohols, Ketones, Glycol ethers, Glycol esters, etc.

2) Hydrocarbon solvents: Aliphatic and Aromatic hydrocarbons.

3) Halogenated solvents: Chlorinated hydrocarbons.

Dryland agriculture refers to cultivation of crops entirely under natural rainfall without irrigation. It is a form of subsistence farming in the regions where deficit of the soil moisture retards the growth of water consuming crops like rice (Oryza sativa), sugarcane etc. Dryland areas are characterized by low and erratic rainfall and no assured irrigation facilities. Dryland agriculture is important for the economy as most of the coarse grain crops, pulses, oilseeds, and raw cotton are grown on these lands. Dryland areas receive annual rainfall between 500 and 1200 mm.

Types of Dryland Agriculture

Depending on the amount of rainfall received, dryland agriculture has been grouped into three categories:

Dry farming: It is production of crops without irrigation in areas where annual rainfall is less than 750 mm. Crop failures are more frequent under dry farming condition owing to prolonged dry spells during crop period. The growing season is less than 200 days. It is generally practiced in arid regions of the country

In order to provide for the establishment of an effective system for protection of plant varieties, plant breeders’ rights and the farmers’ rights and to encourage the development of new varieties of plants it has been considered necessary to put in place a legal protection mechanism the Protection of Plant Varieties and Farmers’ Rights Act, 2001 has been enacted in India (www.plantauthority. gov.in). For the purposes of this Act, Protection of Plant Varieties and Farmers’ Rights Authority has been established and is located at NASC Complex, DPS Marg, Opp - Todapur, New Delhi-110 012.

Early identification of crop diseases enables timely management interventions and can reduce losses in agricultural production. This chapter investigates the use of artificial intelligence and sensor data for detecting crop diseases at an early stage. It introduces the fundamentals of crop disease detection and reviews AI techniques applicable to agricultural monitoring. Sensor technologies provide continuous information about crop and environmental conditions, while AI models analyze these data to identify disease-related patterns. The chapter presents an AI- and sensor-based framework, including implementation and experimental procedures, followed by performance analysis. Challenges and limitations are examined, together with future research directions aimed at improving accuracy, reliability and real-time disease monitoring.

Forecasting and warning systems for pests and diseases play an important role in Integrated Pest Management (IPM) and in farm advisory work. Forecasting pest outbreaks during the growing season and applying control measures only when necessary is very important in conventional farming, but also in organic farming, to increase profitability while, at the same time, minimising negative effects on flora, fauna, and groundwater (Sigvald, 2014). Number of disease-warning systems have been developed and validated for dozens of crops. A few are in wide use by growers, and represent encouraging success stories in IPM. Most warning systems, however, have not made the transition from scientific validation to real-world application. his implementation shortfall is not limited to diseasewarning systems, but also characterizes most other types of agricultural decision support systems (DSSs), whose rate of grower adoption is widely perceived as disappointing (Mark L. Gleason et.al., 2008). 

Eastern blotting is a technique to detect protein post translational modifications (PTM) and is an extension of the biochemical technique of western blotting. There are multiple versions of the method, but in general proteins or lipids are blotted from one or two-dimensional SDS-PAGE gel on to a PVDF or nitrocellulose membrane are analyzed for post-translational protein modifications using probes that may detect lipids, carbohydrate, phosphorylation or any other protein modification. Eastern blotting should be used to refer to methods that detect their targets through specific interaction of the PTM and the probe, distinguishing them from a standard Far-western blot. The most common application is lectin blotting, however, a variety of other probes can be used for detecting different PTMs.

History and Multiple Definitions

Definition of the term Eastern blotting is somewhat confused due to multiple sets of authors dubbing a new method as Eastern blotting, or a derivative therof. All of the definitions are a derivative of the technique of Western blotting developed by Towbin in 1979. The current definitions are summarized below in order of the first use of the name, however all are based on some earlier works. In some cases, the technique had been in practice for some time before the introduction of the term.

Definition of terminologies and concept

Variables: Variables are the characteristic, number or quantity which increases or decreases over time and takes different values in different situations. Variables are those, whose value varies according to the situations. For example, maize yield, Stover, fertilizer, labor, land, pesticides, etc.
 
Dependent variable: Variables whose value is dependent on independent variables. These variables take different values only in response to independent variables. Yield of maize is dependent on amount fertilizer, fertilizer application methods, labor, pesticides etc. So in this case maize yield is dependent variables while amount fertilizer, fertilizer application methods, labor, pesticides are independent variables. However in other situation, maize yield can be used as independent variables also.

Experimental mutagenesis is an important unconventional source to produce mutations in high frequencies in cultivated plants. It makes possible the achievement of desired breeding objectives or atleast decisive progress in this direction through selection of mutants. By establishing extensive collection of mutants based on productive varieties excellent sources of valuable material for theoretical studies and for breeding work can be provided. The induced mutagenesis helps in diversifying varieties and in internal rectification. The simultaneous realization of different breeding objectives may be possible through induced mutagenesis especially in leguminous crops.

Stubbe (1959) stated that mutation breeding as a modern method of plant breeding has proved its justification. It mutation breeding, basic information is a prerequisite on the type of spectrum of induced mutations and relative effectiveness and efficiency of different mutagens.