Preface Molecular diagnostics is an emerging and rapidly evolving area that has revolutionized our approach towards therapy and disease management. Molecular diagnostic tools are used to analyze the presence of nucleic acids or protein associated with a specific health condition. Following diagnosis molecular diagnostic tests can not only provide information about the underlying molecular mechanism behind any disease, but also help in guiding appropriate treatment regime. Now a day’s molecular diagnostic is routinely used by laboratories and hospitals to provide faster and accurate disease management. Various molecular diagnostic assays such as PCR, RT-PCR, Sequencing, FISH can detect biomarkers responsible for a particular disease. With the help of these assays it has become possible to diagnose various chronic diseases such as cancer in its early stage of progression. As molecular diagnostics has proved to be very useful in human health care, it is very important to have basic idea about various tools and techniques used in routine diagnostic practices. Keeping this in view, the present book has been written in such way that it can serve well as laboratory handbook for laboratory personnel in diagnostic laboratories working in the field of molecular pathology. The entire content of the book has been divided into 13 chapters. Starting with an introductory chapter, most parts of the book explains various tools and assays that are routinely used in diagnostic laboratories. Few dedicated chapters such as chapter 2 on laboratory mathematics will help reader to prepare common laboratory solutions while chapter 3 gives idea about basic instrumentation that a standard laboratory should be equiped with to perform molecular diagnostic tests. The book thoroughly explains different nucleic acid extraction protocols that enable isolation of high quality DNA and RNA from human blood or tissue samples. Basic mechanism of some of the very sensitive molecular diagnostic assays which are cost effective, less time consuming and that produces high quality test results are explained in a very simple and understandable language. These tools can easily be applied in diagnosis of different cancer. The last chapter of the book provides insight into the quality control strategies of a molecular diagnostic laboratory. These chapters will help molecular biologists and laboratory technicians to understand and apply these guidelines to ensure safe and consistent work environment.

Molecular biology is a branch of science that deals with understanding the biological activities at the molecular level. Molecular biologists are involved in understanding the concepts behind various intra and intercellular interaction of molecules, contributing to the knowledge about interrelationship of DNA, RNA and protein synthesis, i.e., how genes are transcribed into RNAs and how RNA is translated into protein, the phenomenon which is commonly known as “Central Dogma”stated by Francis Crick in 1956. “The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid”-Francis Crick.

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.

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.

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.

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.

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.

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.

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

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

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.

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.

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.

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.