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Biomedical Technology and Devices Handbook 1140_S03.fm Page 1 Monday, July 14, 2003 9:17 AM III Biological Assays Contributions of Technology to the Biological Revolution Almost all disease states can be ultimately linked to some sort of dysfunction at the cellular or molecular level. The ability to identify abnormalities in the cellular content of diseased tissues has expanded clinical medicine. New technologies to manipulate these processes are emerging every day. At the frontline of this technology is the variety of assays used in clinical laboratories to examine the basis of human disease. The development of these tools began almost immediately following the identification of cells as the basic building blocks of living beings. Microscopic assessment of tissues and cells, combined with various staining techniques, has greatly enhanced disease diagnosis. Many of these techniques have been fine- tuned to the point of almost total automation. As the biological revolution and information age continue to mature, the ability of clinicians to obtain accurate diagnoses will continue to increase. This section includes the following chapters: 13. Molecular Biology Techniques and Applications 14. Theoretical Considerations for the Efficient Design of DNA Arrays 15. Biological Assays: Cellular Level 16. Histology and Staining 17. Radioimmunoassay: Technical Background © 2004 by CRC Press LLC 1140_book.fm Page 1 Wednesday, July 9, 2003 8:46 AM 13 Molecular Biology Techniques and Applications CONTENTS 13.1 Introduction 13.2 Isolation of Nucleic Acids DNA • RNA 13.3 Analysis of Nucleic Acid Hybridization-Based Techniques • Polymerase Chain Reaction • High-Throughput Techniques 13.4 Applications Lidia Kos Cardiovascular • Cerebral/Nervous • Orthopedic • Muscular • Sensory • Digestive • ENT • Pulmonary • Lymphatic Florida International University • Reproductive Roman Joel Garcia Acknowledgments Florida International University References 13.1 Introduction The development and implementation of molecular biology techniques have had a major impact in both basic and applied biological research. Since its inception in the early 1950s, molecular biology has matured as a science. In the last 10 years many of the tedious, repetitive techniques have been automated; time- consuming multistep procedures have been converted into kits; high quality genomic and cDNA libraries are now commercially available, and improvements in the quality of reagents and enzymes have immensely benefited all nucleic acid manipulations. All molecular biology techniques, from the most basic to those more sophisticated and novel, rely on the fundamental processes of molecular biology, DNA replication, transcription, and translation. DNA is a polymer of nucleotides organized as a double-stranded helix. The strands are antiparallel and the nitrogenous bases (adenine [A], cytosine [C], thymine [T], and guanine [G]) that constitute the nucle- otides are held together by hydrogen bonding. DNA replication is semiconservative, meaning that each parent strand acts as a template for the synthesis of a new strand. Therefore, the resulting pair of DNA helices each contains one parent strand and one newly synthesized daughter strand. During replication, the enzyme DNA polymerase catalyzes the addition of nucleotides to the 3¢end (free 3¢ hydroxyl group) of each growing strand, thereby synthesizing these two strands in the 5¢Æ3¢ direction. The substrates for this polymerization are deoxyribonucleoside triphosphates, which are hydrolyzed as they are added to a given growing strand. The sequence of added nucleotides is dependent upon complementary base pairing with the template strands (As with Ts, and Gs with Cs). © 2004 by CRC Press LLC 1140_book.fm Page 2 Wednesday, July 9, 2003 8:46 AM 13-2 Biomedical Technology and Devices Handbook The genetic information carried in DNA is expressed in two basic steps: DNA is first transcribed to RNA, and then RNA is translated into protein. In a given region of DNA, only one of the two strands can act as a template for transcription. Transcription from the DNA template strand occurs in a 5¢Æ3¢ direction and it is catalyzed by RNA polymerase (RNA polymerase II in eukaryotes) using riboses as substrates. RNA is generally single stranded, and it contains uracil pyrimidines instead of thymine bases. After transcription, the RNA molecule travels from the nucleus into the cytoplasm where translation occurs, thus its designation as “messenger RNA” (mRNA). During translation, amino acids are linked in an order specified by nucleotide triplets (codons) grouped within the mRNA. This is accomplished by an adapter transfer RNA (tRNA), which has an anticodon complementary to the mRNA codon, and which binds its corresponding amino acid. Specifically, a family of activating enzymes (aminoacyl-tRNA synthetases) attaches the amino acids to their appropriate tRNAs and subsequently form charged tRNAs. Translation is initiated when the charged tRNAs form a complex with the mRNA and ribosome. The ribosome moves along the mRNA one codon at a time, elongating the nascent polypeptide from the amino terminus toward the carboxyl terminus. After translation, proteins can undergo further modifi- cation by proteolysis, glycosylation, and phosphorylation. This chapter provides an overview of the most commonly used and emerging molecular biology techniques, with a focus on the isolation and analysis of nucleic acids, especially as used in the examination of differences in gene expression. Changes in mRNA levels account for morphological and phenotypic manifestations and are good indicators of cellular responses to environmental signals. Establishing the expression patterns of multiple genes can elucidate the biochemical pathways and regulatory mechanisms that govern cell physiology. These types of studies also have the potential to identify the causes and consequences of diseases such as cancer, and aid in the development of drugs and their possible targets (Lockhart and Winzeler, 2000). Although this chapter does not discuss the wide variety of protein assay techniques, such as western blots, two-dimensional gels, protein or peptide chromatographic separation and mass spectrometric detection, the use of specific protein-fusion reporter constructs and colorimetric readouts, or the char- acterization of actively translated polysomal mRNAs, these peptide-based protocols are the basis for several novel areas of research and they have recently garnered substantial attention (Pandey and Mann, 2000). A testament to this focus is reflected in the newly formed international alliance of industry, academic, and government members (HUPO: Human Proteome Organization) geared to drive a global effort aimed at determining the structure and function of all human proteins (Kaiser, 2002). For readers who seek a wider range of exposure to molecular biology techniques or for those who require detailed laboratory protocols, such information can be found in the references cited within the sections of this chapter, or the literature within Sambrook and Russell (2001) and Ausubel et al. (2002). 13.2 Isolation of Nucleic Acids 13.2.1 DNA The success of most molecular biology techniques depends on the quality of the starting nucleic acid preparation. In the case of DNA, the preparation should be free of contaminating RNA, proteins, lipids, and other cellular constituents that may disrupt the activity of the variety of enzymes used in these techniques. It should also be free of DNA nucleases, which can nick and degrade high-molecular-weight DNA. The isolation method also has to be gentle enough to forego levels of mechanical shear stress that can fragment large genomic DNA. The purification protocol generally starts with the use of a buffer containing one or several detergents (i.e., sodium dodecyl sulfate [SDS], Nonidet P-40, Triton X-100) in order to lyse the cells and disassociate the proteins from DNA. Incubation with proteinase K at high temperatures (56–65o) is used when further deproteinization is required. Following the proteinase K treatment, residual protein and contaminating lipids can be effectively removed by extraction with phenol and chloroform. Contaminating RNA can be degraded with DNase-free RNase, followed by a chloroform extraction. Pure DNA is then obtained by a simple precipitation with isopropanol or ethanol. Organic © 2004 by CRC Press LLC 1140_book.fm Page 3 Wednesday, July 9, 2003 8:46 AM Molecular Biology Techniques and Applications 13-3 extractions can be avoided by employing a prolonged proteinase K incubation step followed by the addition of a saturated NaCl solution to precipitate proteins. Isolated DNA can be subsequently quantified in a spectrophotometer and evaluated for quality by agarose-gel electrophoresis. One of the procedures that requires high quality pure DNA is the construction of a genomic DNA library. After isolation, genomic DNA is digested into small fragments with restriction endonucleases that cleave DNA at specific sites, defined by a sequence of bases (restriction site). A given fragment is ligated into a self-replicating vector that enables it to be maintained and propagated within bacterial cells such as Escherichia coli or yeast cells such as Saccharomyces cerevisiae. Such a library may be stored as a permanent source of representative sequences of a particular organism. They can then be used for screening to isolate a particular sequence of interest, or to determine the location and order of sequences in the genome from which the library was constructed (physical mapping). 13.2.2 RNA There are various
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