eChapters

Reagents

Chloroform

EDTA, 0.5 M

Ethanol, absolute

Isoamyl alcohol

Phenol

Phosphate Buffered Saline (PBS), 1X

Proteinase K

RNAse A

Sodium dodecyl sulfate (SDS) solution, 10%

Individuals in a population can vary considerably on physical, cognitive, and behavioural traits. Examples include height, weight, intelligence, personality, etc. Individual differences may arise from variation in genes, environmental experiences, or a combination of both. It is generally accepted that individual differences in most traits are due to both genetic and environmental factors. To the extent that a trait is influenced by genetic (heritable) effects, phenotypic similarity is correlated with genetic relatedness.
The degree of resemblance among relatives allows for the estimation of genetic variance as well as heritability of a trait. We will now focus on how phenotypic correlation of relatives can be used to estimate the relative magnitude of genetic and environmental influences on trait variation
Partitioning of Phenotypic Values
We previously introduced the general model of Y = G +E where Y is the trait value, G is the genotypic value, and E is the environmental deviation that is assumed to have a mean of 0 so that E(Y ) = E(G). If we assume that the genetic and environmental components are independent, we have that the variance of Y is Var(Y ) = s 2 Y = s 2 G +s 2 E .
We previously focused on the decomposition of the genetic variance s 2 G into additive and dominance components, i.e., G = A+D

Q.1.    In what sense the analysis of covariance is an extension of ANOVA ?

    Answer
    
    The analysis of covariance is an extension of ANOVA in the sense that it covers the case where observations are taken on more than one variable from each experimental unit. The interest centers on the study variable y which depends upon on auxillary variable x.

Reagents

Chloroform

EDTA, 0.5 M

Ethanol, absolute

Is amyl alcohol

Phenol

Proteinase K

RNase A

Sodium acetate, pH 5.2

Sodium chloride, 5 M
Sodium thiocyanate (NaSCN), 1 M

TE Buffer (Tris-EDTA), pH 7.4

Tween 20

Xylene

To address the present day problems in agriculture and horticultural sectors and also to equip the graduating and postgraduate agricultural students, the ICAR has been continuously striving to bring necessary reforms for quality enhancement in agricultural education. The Council has appointed National Core Group and BSMA Committee for revision and restructuring of Post graduate and Doctoral syllabi in consultation with all the stakeholders to meet the challenges and harness opportunities in various disciplines of agriculture and allied sciences. It has been felt that a paradigm shift is necessary in academic regulations to comply with various provisions of National Education Policy-2020. The syllabus of Masters’ and Doctoral programs was restructured and new courses were introduced.

Principle

Restriction digestion is one of the most common reactions performed in molecular biology. Restriction endonucleases (RE) are also known as restriction enzymes, which recognize short, specific DNA sequences. There are three types of RE: Type-I, Type-II and Type-III. Type II RE, most commonly used for DNA analysis and genetic engineering, cleave double-stranded DNA (dsDNA) at specific sites within or adjacent to their recognition sequences. All Type II RE recognises a unique nucleotide sequence at which it cuts a DNA molecule and nowhere else. The recognition sequence is often a six base pair palindromic sequence (the top DNA strand from 5' to 3' is the same as the bottom DNA strand from 5' to 3'), but others recognize four or even eight base pair sequences. Many RE will not cut DNA that is methylated on one or both strands of the recognition site, although some require substrate methylation.

REs can also differ in the way they cut the DNA molecule. Some enzymes cut in the middle of the recognition sequence, resulting in a flush or “blunt end”. Other enzymes cleave in a staggered fashion, resulting in DNA products that have short single-stranded overhangs (usually two or four nucleotides) at each end. These are often called “cohesive ends”, as these single-stranded overhangs could potentially come together again through complementary base-pairing, as shown below:

Principle

Since the entire DNA sequence of the bacteriophage is known, we know exactly where a restriction enzyme will cleave the viral DNA and the size of the DNA fragments generated. This restriction enzyme digest will create a pattern of specific DNA fragments that will serves as a “DNA ladder”.

Identify main reasons and purpose of curriculum development and key areas needed by stakeholders for curriculum change. This can be done by adopting participatory methods like Focus group discussion/ field level workshops. Carry out stakeholder analysis and identify specific potential areas suggested by stakeholders to be included in the curriculum for better performance at the f ield level and their likely role in the process. In order to encourage greater stakeholder involvement invite stakeholders such as staff and students from various farm universities, field extension workers and farmers.

Principle of DNA Ligase Mechanism

The process of connecting two pieces of DNA together is called ligation, and is catalyzed by an enzyme called ligase. The ligase used most often in molecular biology is derived from the T4 bacteriophage, and uses ATP to supply the energy necessary for the reaction. In addition to ATP, T4 ligase requires DNA with a 5´-phosphate group and free 3´-hydroxyl groups.

Applications

Ligation of blunt or cohesive-ended DNA fragments.

Repair of nicks in double-stranded nucleic acids.

Principle

Cloning experiments would be very difficult if we do not screen those very few cells that took up the recombinant plasmid DNA, as the transformation efficiency is very low as stated before (usually less than 1 in 10⁵ cells). Hence it is necessary that there should be a selection mechanism (i.e. conditions where cells grow if they express a gene, but die if they do not) to sort out which are transformed cells and which are not make the job easy.

A commonly used selection mechanism is antibiotic resistance. If the E. coli strain used is not resistant to the antibiotic, but the plasmid DNA contains a gene coding for resistance, then only the cells that have taken up the plasmid will be able to grow. Most commonly used plasmids contain a gene for resistance to ampicillin; only cells that have taken up the plasmid will be able to grow and form colonies.

Note: Uptake of plasmid DNA is rare; hence each group of cells visible on a plate represents the offspring of a single cell that will contain a single molecule of plasmid DNA.

The tissue/cell culture lab has the requirement of maintaining an aseptic environment. There are several aspects to the design of good tissue culture facilities. Ideally, cell culture work should be conducted in a single-use facility. Therefore, cell culture lab unit should be separated from the rest of the lab area. It can be either separate lab or a closed partition in a big lab. All new material should be handled as ‘quarantine’ material in a separate area, and materials known to be free of contaminants should only be allowed inside the culture room. In addition, the work surfaces should be thoroughly cleaned and disinfected between activities.

For most of the work involving non-infectious primary culture and cell lines, the laboratory should be designated to at least BSL2 facility. The location of the Biosafety cabinet is the most important point to consider. When operated correctly, the cabinet will provide a clean working environment, whilst protecting the operator from aerosols. Environmental monitoring with Tryptone Soy Broth agar settle plates inside the biosafety cabinet is a good indicator of the cleanliness of a cabinet. There should be no growth of bacteria and fungi after 3-5 days of incubation.

The global demand for high producer animals is rising, and cattle producers are looking for ways to improve productivity and efficiency. One method for developing cattle with enhanced traits is crossbreeding – combining the desirable qualities of two or more cattle breeds into one animal.
Crossbreeding allows cattle producers to take advantage of breed differences and heterosis while managing unwanted traits.
Implementing an effective breeding strategy can help cattle producers select for optimum performance in their herd.
Benefits of Crossbreeding Cattle
Crossbreeding offers commercial beef producers several advantages compared to using only straight-breed animals. Some key benefits include:
1. Heterosis – Crossbred animals exhibit hybrid vigour or heterosis, which is the increased performance for traits like growth rate, fertility, and longevity compared to purebred animals.
The heterosis effect results from the increase in heterozygosity or genetic diversity from combining divergent breeds. Heterosis levels are highest in first-cross animals (the initial cross between two pure breeds.
2. Complementarity – An additional advantage of crossbreeding is combining the strengths of two or more breeds to produce an animal with the ideal blend of traits.
For example, crossing a breed known for high intramuscular fat and marbling, like Angus, with a lean breed that excels in meat yield and growth, like Charolais, can result in an animal with both good marbling and leanness.
3. Breed Complementation – Crossbred animals also benefit from breed complementation. This occurs when the strengths in one breed offset or complement the weaknesses in the other breed. For example, British maternal breeds tend to be lower in milk production but exhibit excellent maternal behaviour

Domestic livestock plays a major role in meeting the food requirement of human beings. Especially with dwindling resources, improvement in production efficiency is primary to meet the ever-increasing demand for animal protein. The role of an animal breeder is to bring about a sustained improvement in the quality and quantity of such products. Over the years, there has been an enormous improvement in the production ability of livestock and statistical analysis has played a predominant role in the application of principles of animal breeding to improve the productivity of animals. The combination of genetics and statistics developed into a separate branch of science called biometry.

The Madrid System for the International Registration of Marks is governed by two treaties. The first one is he Madrid Agreement, which was concluded in 1891 and revised at Brussels (1900), Washington (1911), The Hague (1925), London (1934), Nice (1957) and Stockholm (1967), and amended in 1979. The other one is the Protocol related to this agreement which was concluded in 1989. The aims of this protocol are to make the Madrid system more flexible and more compatible with the domestic legislation of certain countries or intergovernmental organizations that had not been able to accede to the Agreement. 

Working Principle: An autoclave is a sealed device that creates a different environment from the outside world under extraordinarily high pressure (Auto + clave = automatic locking). It uses moist heat or saturated steam under pressure to kill microorganisms and heat-resistant endospores. Its function is compared to that of a pressure cooker, i.e., steam is used to create a high pressure environment. However temperature of autoclave or steam sterilizer is generally maintained at 121°C for 15-20 minutes that is achieved by raising the pressure inside the device. This high pressure increases the boiling point of water and therefore raises the temperature that is too high for the microbes to survive, so used for sterilization.

Types of Autoclave cycle

1. Liquid cycle: Liquids take time to sterilize as they have high heat capacity. It is based on the principle of pressurized steam at 15 psi, 121°C temperature for 20 minutes. It has latent heat, which is 7× heat 100°C. Liquids and biological waste require slow exhaust to prevent the boiling over of super-heated liquids. This excess temperature is enough to hydrolyze the proteins of bacteria, viruses, or any other contaminant and therefore kill it. However, wet heat is seven times more intense than dry heat, so it requires less temperature than the dry heat cycle of the autoclave.

2. Dry Cycle: Here, steam is the sterilization medium, which penetrates inside the load and makes contact with all the surfaces, so a fast exhaust cycle is used. It has a high temperature of 160°C for 60 minutes.

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. 

It is the most significant international convention in the field of protecting copyright. The Berne Convention deals with the protection of works and the rights of their authors. It is based on three basic principles and contains a series of provisions determining the minimum protection to be granted, as well as special provisions available to developing countries that want to make use of them. The Berne Convention, signed on 9 September, 1886, was revised at Paris in 1896 and at Berlin in 1908, completed at Berne (Switzerland) in 1914, revised at Rome in 1928, at Brussels in 1948, at Stockholm in 1967 and at Paris in 1971, and was amended in 1979.

Understanding Immunogenetics
The study of genetic basis of the immune response is known as immunogenetics. The term was introduced with the discovery of ABO blood groups and were first demonstrated through the existence of “natural” antibodies i.e., isoantibodies. The broad field of immunogenetics include study of normal immunological pathways as well as identification of genetic variations that result in immune defects, which may facilitate detection of new therapeutic targets for immune diseases. Hence, understanding and subsequent manipulation of host immune response (immunomodulation) is the most precise and effective tool to reduce disease incidences and nullify the limitations associated with antibiotic treatment or vaccination. Therefore, breeding for disease resistance has gained considerable attention from researchers in the recent past.
Immune Response Genes
Often it was observed that individuals respond differently to same infectious agent. A possible explanation may be the genetic variability between them. Indeed, many studies have looked for associations between genes involved in immunity and disease outcome and it has been found that certain immune response (Ir) genes play the crucial role. This concept was discovered in the mid-1960s and this discovery introduced an apparently new level of antigen recognition whose diversity and specificity had to be explained in addition to those of familiar immunoglobulins. Hence immune response (Ir) genes were defined as antigenspecific genes that control the ability of an animal to raise an immune response, either humoral or cellular to a particular antigen .

What is primary cell culture?

In vitro culture of cells freshly obtained from the tissue is called primary cell culture. Unlike that of cell line, primary cell culture grows for a limited period of time. Examples, are primary mammary epithelial cells, primary hepatocyte culture.

Aim: To prepare the primary cell culture from chicken embryos.

Materials required for the primary cell culture

• 70% alcohol

• Cotton swabs

• Small beaker 20-50 ml

• Forceps

• Straight and required curved,

• 9 cm Petri dishes

• 11 day embryonated eggs

• Balanced Salt solution (BSS)

• 250 ml conical flask

• Magnetic centrifuge tubes

• Haemocytometer

• Growth medium with serum

• Culture flasks

What is keratinocyte?

The epidermal layer consists of keratinocytes, melanocytes, Langerhans cells, and Merkel cells. Their major function is to produce Keratinocytes which are the most abundant cells in the epidermis, keratin protein. Keratinocyte synthesizes major structural components of the epidermal barrier through a programmed process of differentiation. They are present in the innermost layer of the epidermis (Eckert et al?, 1989). The epidermis is composed of several layers, stratum corneum (horny layer), stratum lucidum (clear layer), stratum granulosum (granular layer), stratum spinosum (prickle cell layer), and stratum basale (basal layer), from the outermost to the deepest layer respectively. The process of maturation and movement of the keratinocytes from the stratum basale to the stratum corneum is what we call epidermal differentiation.

Procedures for isolation of canine karatinocytes

When studying skin cells, isolating keratinocytes from tissue samples, such as skin biopsies, is a critical initial step. Here is a general guide on how to isolate keratinocytes: