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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.
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 the discussion of the techniques for isolating and analyzing nucleic acids in Chapters 63 and 64 . Chapter 65 will focus on genomes and their variants, as well as massively parallel methods, while Chapter 66 discusses clinical genome sequencing in depth. The clinically important subdivisions of molecular diagnostics are then reviewed and include microbiology in Chapter 67 , genetics in Chapter 68 , solid tumors in Chapter 69 , and hematopoietic malignancies in Chapter 70 . Chapters 71 and 72 are devoted to the molecular diagnostic analysis of circulating tumor cells and circulating nucleic acids. Finally, pharmacogenetics is the focus of Chapter 73 .
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. , 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.
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 cytosine and the pivotal x-ray crystallography results produced by Rosalind Franklin and Maurice Wilkins. ,
Molecular biology has historically traced its beginnings to the first description of the structure of DNA by James Watson and Francis Crick in 1953. , 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.
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. , , One of those properties was the replication of DNA, which was shown to be semiconservative by Meselson and Stahl in 1958. At the same time, DNA polymerase, which replicates the DNA, was discovered by Arthur Kornberg. 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. 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. , Another finding showed that the eukaryotic gene structure was composed of alternating non–protein-encoding introns and protein-encoding exons. , Along with the discovery of the basic biology of genes and their expression, many important techniques were invented. For example, the isolation of restriction enzymes and DNA ligase allowed for the construction of recombinant DNA, which could be transferred from one organism to another, leading to the cloning of DNA 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. DNA sequencing technologies were invented , 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 1986 and microarray technology in 1995, became methodologic foundations for molecular diagnostics.
Whether it is a bacterium, virus, or eukaryotic cell, the genetic material located in an organism dictates its 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. 62.1 and 62.2 ). DNA in human cells is wrapped around histone proteins and packaged into nucleosome units, which are compacted further to form chromosomes ( Fig. 62.3 ). 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 set of chromosomes, 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.
DNA is a rather simple molecule with a limited number of components compared to those of proteins. DNA is composed of a deoxyribose sugar, phosphate group, and four nitrogen-containing bases. Deoxyribose is a pentose sugar containing five carbon atoms that are numbered from 1′ to 5′, starting with the carbon that will be attached to the base in DNA and progressing around the ring until the last carbon that is not part of the ring structure. The bases consist of the purines, adenine and guanine and the pyrimidines, cytosine and thymine; an additional base, uracil, replaces thymine in RNA. A basic building block is the nucleotide, which consists of a deoxyribose sugar with an attached base at the 1′ carbon and a phosphate group at the 5′ carbon. The triphosphate nucleotide is the building block for making newly synthesized DNA. Newly synthesized DNA forms a polynucleotide chain that connects the individual nucleotides through the 5′ and 3′ carbons of each deoxyribose sugar via phosphodiester bonds.
DNA is double stranded, and the two strands bind to one another through hydrogen bonds between the bases on each strand. Hydrogen bonding is augmented by hydrophobic attraction (stacking) between bases on adjacent rungs of the DNA ladder. Both hydrogen bonds and base stacking are not covalent, but are weak bonds that can be broken and reestablished. This important property is exploited by many of the methods that are used in molecular diagnostics. The composition of DNA is equal quantities of guanine and cytosine and equal quantities of adenine and thymine, because, in general, guanine binds to cytosine and adenine binds to thymine. , There are two hydrogen bonds between adenine (A) and thymine (T) and three hydrogen bonds between cytosine (C) and guanine (G), and because of this difference in the number of hydrogen bonds, separating a guanine-cytosine (G-C) pair takes more energy than an adenine-thymine (A-T) pair (see Fig. 62.1 ).
Each of the two DNA strands is formed by an alternating phosphate sugar backbone that starts at the 5′ phosphate and ends at a 3′ hydroxyl group with the complementary bases binding to one another between the two phosphate sugar backbones. Each strand is therefore a polar opposite of the other (see Fig. 62.2 ). When the two strands are bound to one another they progress in opposite 5′ to 3′ directions in an antiparallel configuration. By convention, the DNA sequence is denoted in a 5′ to 3′ direction. As discussed later, both the replication of new DNA and the transcription of DNA to RNA progress in the 5′ to 3′ direction. In addition, the conversion of RNA to protein, a process called translation, proceeds from the 5′ end of the RNA to the 3′ end. The combination of the base pairing and the directionality of the two DNA strands allows for the deciphering of the DNA sequence on one strand of DNA when the other complementary strand sequence is known.
Double-stranded DNA in living cells is generally found as the right-handed B-DNA helical structure, which has specific dimensions. Each turn of the helix is 3.4 nm long and consists of 10 bases. The DNA sugar-phosphate backbone is on the outside of the helix, and the bases of each strand are inside bound to their complement on the other strand by hydrogen bonds. Other conformational structures of DNA occur, mostly associated with DNA sequences that are repeated. These non-B DNA forms include a left-handed Z-form, A-motif, tetraplex G-quadruplex, i-motif, hairpin, cruciform, and triplex and are abundant in the human genome because a large percentage of the genome contains various repeats. Non-B DNA is associated with many biological processes, including transcriptional control. However, these structures also can create genetic instability, which can lead to various diseases such as neurologic disorders.
The composition of RNA is similar to that of DNA because it contains four nucleotides linked together by a phosphodiester bond, but with several important differences. RNA consists of a ribose sugar with a hydroxyl group at the 2′ carbon instead of the hydrogen atom in DNA. The bases attached to the ribose sugar are adenine, cytosine, and guanine, but not thymine because RNA uses another pyrimidine—uracil—as a substitute for thymine.
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