Molecular pathology is based on the principles, techniques, and tools of molecular biology as they are applied to diagnostic medicine in the clinical laboratory. These tools were developed in the research setting and perfected throughout the second half of the 20th century, long before the Human Genome Project was conceived. Molecular biology methods were used to elucidate the genetic and molecular basis of many diseases, and these discoveries ultimately led to the field of molecular diagnostics. Eventually the insights these tools provided for laboratory medicine were so valuable to the armamentarium of the pathologist that they were incorporated into pathology practice. Today, molecular diagnostics continues to grow rapidly as in vitro diagnostic companies develop new kits for the marketplace and as the insights into disease gained by the progress of the Human Genome Project develop into laboratory tests.
Molecular pathology is a natural extension of anatomic and clinical pathology. As molecular research identifies the most fundamental causes and markers of disease, clinical testing is moving to the nucleus and its genetic material. Underlying mutations responsible for genetic diseases, including cancers, are being discovered and used in molecular diagnostic tests. Some have become routine tests in molecular pathology and are described in detail elsewhere in this book.
In this chapter, fundamental and more advanced molecular biology techniques, as practiced in the molecular pathology laboratory, are reviewed. The entire field of molecular pathology is relatively new, having begun in the 1980s and matured through the 1990s. The new century has brought important advances in automated nucleic acid preparation,1-3 polymerase chain reaction (PCR), real-time PCR, and deoxyribonucleic acid (DNA) sequencing. More and more, the underlying biochemistry occurring in laboratory instruments may be invisible to the user. The goal of this chapter is to describe the principles of these methods so that practitioners have adequate information for instrument troubleshooting and test interpretation.
Obviously, gene products, that is, proteins and polypeptides, are molecules and could technically be classified within "molecular pathology." This chapter focuses on nucleic acid diagnostics, including investigation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The field will eventually move beyond genomics to pro-teomics. Proteomics is not addressed in this chapter since clinical proteomics is in its most nascent stages and is in extremely limited practice in molecular pathology today.
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