eChapters

Estimation of variance is primary for any breeding program as all the genetic parameters are dependent on these estimates. The properties of an estimate, their assumptions, type of data, availability of data, and computational requirement make it important to understand the method of estimation of variance components so as to make a wise choice that would suit the breeder’s requirement. This chapter explains the methods of estimation of variance with merits and demerits of each and the reader could use these as a guide to choose the right method of estimation.

The genetic parameters are the basis for any animal breeding program and the important parameters such as heritability, repeatability and genetic correlation are ratios of (co)variances and their reliability depends on the accurate estimation of variance components. Estimates of variance components are used extensively in animal breeding.

The key genetic parameters available with the animal breeder to attain improvement are heritability, repeatability and genetic correlation. This chapter will mainly deal with the theoretical considerations of these parameters.

The goal of an animal breeder is to bring about permanent improvement in different economic traits, either individually (univariate) or simultaneously in multiple traits (multitrait). The phenotypic value available from the animal is a combination of genotypic value and environmental deviation. By permanent improvement in a trait, we mean the improvement in genotypic value, which could be carried over to future generations. The environmental deviations are spatially and temporally specific, and thus will not be the same over generations. The genotypic value, itself is made up of additive effect, dominance deviation and interaction deviation. The additive effect is a value of genes (the sum of the average effect of genes) and hence is more important for the breeder as only the genes are passed on to the next generation.

It is known that the phenotype of an individual is a combination of genotype and environment. Animal breeding analysis is mainly done to partition the genetic part from the environmental part so as to assess the actual genetic worth of the animal. In this process correction for the known environmental part is integral to any animal breeding analysis. In this chapter, a list of common factors affecting important economic traits are described. The analytical part for these effects will be explained in later chapters.

Correction or standardization of data is very important in any animal breeding analysis. Unless the candidates under selection are placed on a common platform, various factors affecting the trait will lead to bias in assessing the performance of the individual with respect to a particular trait. For eg. a lamb born during a favorable season, a lactating cow in its third parity, first lactation yield of a cow with longer age at first calving.

Correlations and regression are important components of animal breeding analysis. The genetic correlation is an important genetic parameter that is useful in the planning of breeding programs involving multiple traits. In this chapter, the basic concepts of correlation and regression including method of estimation, assumptions and test of significance are presented. An example using SPSS software is also given at the end for better understanding.

Basic knowledge of statistics is important to understand animal breeding analysis. In this chapter, the hypothesis testing, principles of experimental design, types of data and probability distribution are described with examples using appropriate software.

Animal breeding data is unique in terms of volume, statistical analysis, type of information required etc. The large volume of data extended spatially and temporally warrants additional tools in the form of databases to handle data and enable accuracy, retrieval, efficient utilization and adaptability to software. This chapter explains the nature of animal breeding data and the importance of databases with examples and explanations.

Animals are raised for some utility including meat, milk, hide, wool etc., and efforts are on to continuously improve the quality and quantity of these products. Some of the important economic traits in this regard were already discussed in earlier chapters. Record maintenance is primary for decision-making with respect to the selection of animals. The details of record maintenance including the format of registers are already explained in chapter 2. Apart from the actual information required for economic traits, pedigree information forms an important part of animal breeding data.

Maintaining the required type of information in the form of records is primary for any type of animal breeding analysis and this chapter explains the importance of records and ways to maintain them in the form of registers or computer databases. The formats for different types of registers to be maintained are also given as a template in appendix. Any breeding plan is formulated to improve the economic traits depending on the utility of species reared and the chapter lists important economic traits of livestock and poultry with definitions and formulae, where ever required.

Basic record maintenance is a must for any animal breeding program. Every farm or breeding program is expected to maintain records in the form of registers or computerized databases. The goal of animal breeding is to select mates with the objective to improve economically important traits. In this chapter, the most common economic traits in different species of livestock are described. Even though the list is not exhaustive the basic economic traits that have been worked for improvement are explained. Breeders also explore new composite traits which are a combination of these base traits.

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.

Molecular diagnostics has witnessed groundbreaking technological advances in recent times, starting from automated sample preparation to real time amplification technology. Possibility to develop and run assays for ever increasing number of clinical queries has become effortless due to the technological advances. However, the state of the art in quality assurance and quality control practices for molecular diagnostics has seldom been up to the expectations if one considers accuracy for once in a lifetime genetic tests or new genetic test targets appearing almost every single day. In the face of such issues, clinical laboratories are struggling to develop appropriate quality assurance programs for the molecular diagnostic tests they conduct. In addition, legislation and government policies threaten to increase oversight of genetic tests to a level not seen before in laboratory medicine, for example, FDA’s guidance on in vitro diagnostic multivariate index assays (IVDMIA).

To relate patient results to previous test results or to absolute values in clinical practice guidelines, those results need to be comparable across time and methods. This may either be achieved by designing the identical value across methods and test versions or by using reliable and stable conversions. Therefore, the establishment of international standards and reference materials is of paramount importance. Monitoring molecular test system outputs and statistical analysis might provide a good way to obtain such standards; however, such traditional QC (Quality Control) strategies can be slow to take root in molecular diagnostics.This chapter highlights general and specific issues relevant for quality assurance and quality control in the routine molecular diagnostics laboratory and guidelines for designing quality manuals for a molecular diagnostic laboratory.

12.1 Assays for molecular diagnosis of Acute Myeloid Leukaemia (AML)

Acute Myeloid Leukaemia (AML), also known as Acute Myelogenous Leukaemia or Acute Myeloblastic Leukaemia or Acute Granulocytic Leukaemia or Acute non Lymphocytic Leukaemia is an aggressive type of cancer which affects blood and bone marrow. AML is a heterogeneous disorder if one considers its clinical status, therapeutic responses and overall prognosis.¹ This disease called acute because it progresses very fast and myeloid as it originates from myeloid cell line in the bone marrow.

In bone marrow blood cells are made in a controlled way. When AML occurs excess numbers of immature leukaemia cells are produced. Overproduction of leukaemia cells in bone marrow disables bone marrow to make mature white blood cells. These accumulated immature cells are unable to function properly (Inability to fight infection).

Diagnosis: Correct diagnosis is important for a successive treatment for AML. There are several tests available to diagnose AML but not all tests will be used for each person. The following tests may be used to diagnose AML.

11.1 Principle

In situ hybridization techniques involve detection of specific nucleic acid sequences to in morphologically preserved chromosomes, cells or tissue sections. Along with immunocytochemistry, in situ hybridization can correlate microscopic topological information to gene activity at the level of DNA, mRNA or protein. The FISH protocol involves 1) denaturation and hybridisation performed on Day 1 and 2) washing and detection performed on Day 2. DNA of the chromosomes and the probes are denatured and allowed to hybridize overnight on the first day. On day 2, slides are washed to remove unbound DNA sequences followed by detection, counterstaining and mounting of the slides.

10.1 Purpose

To lay down a standard protocol to perform qPCR for quantitative analysis of target genomic sequence.

10.2 Principle

qPCR or quantitative polymerase chain reaction or real-time polymerase chain reaction, is a molecular biology technique to amplify and at a time quantify a targeted DNA molecule and based on the polymerase chain reaction (PCR). It is called real time as the amplified DNA is detected as the reaction progresses and holds an advantage over standard PCR where the reaction is detected at its end. There are two common methods in quantitative PCR to detect the products i) non-specific fluorescent dyes which intercalates with any double stranded DNA ii) sequence-specific DNA probes labeled with a fluorescent reporter consisting oligonucleotides. This reporters helps in detection of the complementary hybridization of the probe and quantifies amount of DNA in cells or tissues.

9.1 Purpose

To lay down a standard protocol to perform RT-PCR for quantitative analysis of target RNA.

9.2 Principle

RT-PCR technology is used to amplify a specific RNA sequence (target sequence) to measure its expression inside the cell. RT-PCR follows the same principle as of normal PCR, but instead of DNA here cDNA is used as template for amplification. Extracted RNA is reverse transcribed to cDNA with the help of reverse transcription enzyme. The cDNA is then subjected for PCR amplification with the help of DNA polymerase, target specific primers, nucleotides and reaction buffer. In molecular diagnosis, RT-PCR is used to detect the expression of oncogenes at the time of diagnosis. It is also used to detect minimal residual disease during treatment

For RT-PCR, the extracted total RNA is first reverse transcribed to cDNA using reverse transcriptase enzyme. Following are the protocols for cDNA sysnthesis and RT-PCR.

Note: cDNA synthesis and RT-PCR can be performed according to the manufacturer protocol from where the specific reagents for the above methodologies are purchased.

8.1 Purpose

To lay down a uniform protocol for the synthesis of c-DNA from total RNA

8.2 Scope

The scope of this Standard Operating Procedure is to lay down a standard procedure for the synthesis of c-DNA from total RNA

7.1 Basic concept

What is DNA sequencing?

The genome of any biological organism is a sequence of nucleotides arranged in a specific manner. The nucleotides are chemically a base which usually exists in 4 different forms; Purine – Adenine (A) and Guanine (G) and Pyramidine – Cytosine (C) and Thymine (T). These four nucleotides make up an entire sequence of Deoxyribose Nucleic Acid (DNA) which is encodes the information of any cell in the form of a gene. The genome, therefore, is the complete set of all genes that is required to build and maintain that organism. DNA sequencing is the process wherein the precise order of these nucleotides along the gene is detected and analysed which includes any method or technology that assists in determining the sequence.

6.1 Designing primers

Designing correct primers are very important to get the expected result in PCR experiment. There are two primers (Forward & Reverse) required for the amplification of the desired DNA fragment. One primer should anneal to the sense strand (oriented to the 5'→3' direction) and another primer should complement the antisense strand (oriented in the 5'→3' direction).

There are various characteristics to be considered to design a good primer including Tm, length, GC content, secondary structure and specificity.

There are commercially available softwares besides free online tools at NCBI (primer blast) for designing PCR primers. In addition, primer sequences published in peer reviewed literature can also be used.

5.1 Purpose

To employ a standard protocol to perform agarose gel electrophoresis.

5.2 Principle

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb. Migration rate of DNA molecule depends on various factors like 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under UV light.

Nucleic acid extraction from clinical samples is the first step to be followed in molecular detection of cancer. Nucleic acids are the starting material for all downstream molecular diagnostic tools. Quality of test results largely depends upon the quality of starting material. Thus utmost importance should be given to the extraction protocols to isolate good quantity and good quality nucleic acids from patient samples.

Basic principle: The basic principle of nucleic acid extraction is to disrupt cell membrane and nuclear membrane to release and precipitate nucleic acid in the solution. Following precipitation,removal of other cellular contaminates such as protein, lipids, polysaccharides etc is performed to obtain a good quantity and quality of nucleic acid.

Molecular diagnostic laboratory set-up has basic requirements of space, laboratory benches and equipments. In terms of laboratory space, minimum four chambers, one each for sample preparation and nucleic acid extraction, PCR reaction mixture preparation, thermal cycling and gel analysis are required. In addition there should be sufficient space for storage of consumables and laboratory wares. In addition, there are some laboratory equipments which are needed in a molecular diagnostic laboratory. In the following section the equipments of a molecular diagnostic laboratory are discussed. The pictures are taken from the instruments present in the laboratory of the author. The purpose of this chapter is to introduce the reader to a molecular diagnostic laboratory. None of the pictures below are meant for promotion of any particular brand or manufacturer.

3.1 UV-VIS spectrophotometer

A spectrophotometer is required for determining the quality and quantity of nucleic acids extracted from a sample. Photometric research applications such as DNA, RNA and protein analysis can be carried out in a UV-Vis Spectrophotometer. There are various units that feature a broad wavelength range (including UV area), path length correction and a fast reading speed. Various instruments also provide an interface that allows quick measurements directly from the instrument or comprehensive easy-to use software for demanding assays.

In any organism and biomolecules (enzyme, proteins, etc.), most biological activities could be seen only within a narrow environmental conditions. Therefore in order to carry out a successful experiment, from cell culturing to DNA sequencing or assessing an enzyme’s activity, one has to pay utmost attention in calculating correct reaction components.

A solution is defined as a homogenous mixture of solute and solvent. In this case the amount solute is less as compared to the solvent. It is not always necessary that solvent is liquid and solute is solid. There are several examples where solute and solvents are found in other physical states such as gas or solids. For example, carbonated water CO₂ is solute and water is solvent; and smoke is solution of different smoke particles (solute) in air (solvent). The concentration of solution is described as amount of solute in a given volume of solvent. Several ways are there to express the concentration of a solution, such as molarity, normality, molality, and ppm. In this chapter we would discuss the general terms used in making solutions and a brief introduction about buffer solutions.