Principles and Applications of Molecular Diagnostics

Nader Rifai,A. Rita Horvath,Carl T. Wittwer,Jason Park

• Publisher: Elsevier
• Year: 2018
• ISBN:
  9780128160626
• Topic: Ciencias físicas
• Subtopic: Química analítica

Principles and Applications of Molecular Diagnostics serves as a comprehensive guide for clinical laboratory professionals applying molecular technology to clinical diagnosis. The first half of the book covers principles and analytical concepts in molecular diagnostics such as genomes and variants, nucleic acids isolation and amplification methods, and measurement techniques, circulating tumor cells, and plasma DNA; the second half presents clinical applications of molecular diagnostics in genetic disease, infectious disease, hematopoietic malignancies, solid tumors, prenatal diagnosis, pharmacogenetics, and identity testing. A thorough yet succinct guide to using molecular testing technology, Principles and Applications of Molecular Diagnostics is an essential resource for laboratory professionals, biologists, chemists, pharmaceutical and biotech researchers, and manufacturers of molecular diagnostics kits and instruments.

• Explains the principles and tools of molecular biology
• Describes standard and state-of-the-art molecular techniques for obtaining qualitative and quantitative results
• Provides a detailed description of current molecular applications used to solve diagnostics tasks

1

Principles of Molecular Biology

John Greg Howe

Abstract

Background

Molecular diagnostics and its parent field, molecular pathology, examine the origins of disease at the molecular level, primarily by studying nucleic acids. Deoxyribonucleic acid (DNA), which contains the blueprint for constructing a living organism, is the centerpiece for research and clinical analysis. Molecular pathology is an outgrowth of the enormous amount of successful research in the field of molecular biology that has discovered over the last seven decades the basic biological and chemical processes of how a living cell functions. The success of molecular biology, as noted by the large number of Nobel prizes awarded for its discoveries, is now used for clinical diagnosis and the development and use of therapeutics.

Content

The following chapters are devoted to describing this field and the specific applications currently being used to characterize and help treat patients with a variety of ailments, including hereditary genetic diseases, cancer neoplasms, and infectious diseases. In this chapter the fundamentals of molecular biology are reviewed, followed by a focus on genomes and their variants in Chapter 2. In Chapters 3 and 4 techniques for isolating and analyzing nucleic acids are discussed. The clinically important subdivisions of molecular diagnostics are then reviewed and include microbiology in Chapter 5, genetics in Chapter 6, solid tumors in Chapter 7, and hematopoietic malignancies in Chapter 8. Chapters 9 and 10 are devoted to the molecular diagnostic analysis of circulating tumor cells and circulating nucleic acids. Finally, pharmacogenetics and identity assessment are the focus of Chapters 11 and 12.

Historical Developments in Genetics and Molecular Biology

Molecular diagnostics would not be possible without the many significant pioneering efforts in genetics and molecular biology. Earlier observations in genetics began with the discovery of the inheritance of biological traits made by Gregor Mendel in 1866 and the observation in 1910 that genes were associated with chromosomes by Thomas Morgan. The initial findings that contributed to determining that DNA was the transmittable genetic material were performed by Griffith in 1928 and Avery, McLeod, and McCarty in 1944.^1,2 The definitive studies, published by Hershey and Chase in 1952, demonstrated that radiolabeled phosphate incorporated into the DNA of a bacteriophage was found in newly synthesized DNA containing bacteriophage instead of radiolabeled sulfur in protein, which showed that DNA and not protein was the genetic material.³

Deciphering the structure of DNA required several crucial findings. These included the observation by Erwin Chargaff that the quantity of adenine is generally equal to the quantity of thymine, and the quantity of guanine is similar to the amount of cytosine⁴ and the pivotal x-ray crystallography results produced by Rosalind Franklin and Maurice Wilkins.^5,6

Molecular biology has historically traced its beginnings to the first description of the structure of DNA by James Watson and Francis Crick in 1953.^7,8 The description of the DNA structure initiated the dramatic increase in the knowledge of the biology and chemistry of our genetic machinery. The impact of the Watson and Crick discovery was so significant that it is considered one of the most important scientific discoveries of the 20th century.⁹

One reason the work of Watson and Crick had such a dramatic impact on scientific discovery was that they not only described the structure of DNA, but hypothesized about many of its properties, which took decades to confirm experimentally.^7,8,10 One of those properties was the replication of DNA, which was shown to be semiconservative by Meselson and Stahl¹¹ in 1958. At the same time, DNA polymerase, which replicates the DNA, was discovered by Arthur Kornberg.¹² Deciphering the genetic code was vital for understanding the information stored in DNA, and cracking the code in 1965 required many scientists, most prominently Marshall Nirenberg.¹³ Additional studies described the transcription and translation processes and uncovered several startling findings. One finding was the isolation of reverse transcriptase, an enzyme that synthesizes DNA from ribonucleic acid (RNA), which demonstrates that genetic information can be transferred in part in a bidirectional manner.^14,15 Another finding showed that the eukaryotic gene structure was composed of alternating non–protein-encoding introns and protein-encoding exons.^16,17 Along with the discovery of the basic biology of genes and their expression, many important techniques were invented. For example, the isolation of restriction enzymes¹⁸ and DNA ligase allowed for the construction of recombinant DNA,¹⁹ which could be transferred from one organism to another, leading to the cloning of DNA²⁰ and the emergence of genetic engineering. The Southern blot method, which identified specific electrophoretically separated pieces of DNA, participated in many discoveries and was one of the first molecular diagnostics methods to be used to test for genetic diseases.²¹ DNA sequencing technologies were invented^22,23 and further advances in these technologies led to the first large biological science research undertaking, the Human Genome Project. Along with DNA sequencing, further technical discoveries, including the polymerase chain reaction in 1986²⁴ and microarray technology in 1995,²⁵ became methodologic foundations for molecular diagnostics.

Molecular Biology Essentials

Whether it is a bacterium, virus, or eukaryotic cell, the genetic material located in these organisms dictates their form and function. For the most part the genetic material is DNA, which is composed of two strands of a sugar-phosphate backbone that are bound together by hydrogen bonds between two purines and two pyrimidines attached to the sugar molecule, deoxyribose, in a double helix (Figs. 1.1 and 1.2). DNA in human cells is wrapped around histone proteins and packaged into nucleosome units, which are compacted further to form chromosomes (Fig. 1.3). There are 23 pairs of chromosomes, two of which are the sex chromosomes, X and Y. Each chromosome is a single length of DNA with a stretch of short repeats at the ends called telomeres and additional repeats in the centromere region. In humans, there are two sets of 23 chromosomes that are a mixture of DNA from the mother's egg and father's sperm. Each egg and sperm is therefore a single or haploid set of 23 chromosomes and the combination of the two creates a diploid set of human DNA, allowing each individual to possess two different sequences, genes, and alleles on each chromosome, one from each parent. Each child has a unique combination of alleles because of homologous recombination between homologous chromosomes during meiosis in the development of gametes (egg and sperm cells). This creates genetic diversity within the human population. If a child has a random DNA sequence change or mutation, the child's genotype is different from that inherited from either of the parents (de novo variant). If the child's genotype leads to visible disease, the child has acquired a different phenotype from the parents.

Human cells have a limited lifespan and die through a process called apoptosis. Therefore most cells replace themselves as they progress naturally through their cell cycle. As a cell moves through phases of the cell cycle, its DNA doubles during the synthesis phase when the double-stranded DNA molecule separates. Each strand of DNA is used as a template to make a complementary strand by DNA polymerase in a process called DNA replication. Eventually during the cell cycle, two cells are created from one during the final mitotic phase.

DNA is composed of genes that code for proteins and RNA. For DNA to convert its store of vital information into functional RNA and protein, the DNA strands need to separate so that RNA polymerase can bind to the start region of the gene. With the help of transcription factors that bind upstream to promoters, the RNA polymerase produces single strands of RNA that are further processed to remove the introns and retain the protein-encoding exons. The mature, processed RNA molecule, the messenger RNA (mRNA), migrates to the cytoplasm, where it is used in the production of protein.

To start the process of protein synthesis or translation, the mRNA is bound by various protein factors and a ribosome, which contains ribosomal RNA (rRNA) and protein. The mRNA-bound ribosome begins to produce a polypeptide chain by binding a methionine-bound transfer RNA (tRNA) to the mRNA's initiating AUG codon or triplet code. The conversion of the nucleic acid triplet code to a polypeptide is accomplished by the tRNA, which contains a nucleic acid triplet code (anticodon) in its RNA sequence that is specific for an amino acid bound to one end of the tRNA molecule. After synthesis, the protein migrates to its functional location and eventually is removed and degraded.

FIGURE 1.1 A, Purine and pyrimidine bases and the formation of complementary base pairs. Dashed lines indicate the formation of hydrogen bonds. (∗In RNA, thymine is replaced by uracil, which differs from thymine only in its lack of the methyl group.) B, A single-strande...