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Cellular and molecular pathology
A discipline that deals with the origins and mechanisms of diseases at their most fundamental level, that of macromolecules such as deoxyribonucleic acid (DNA) and protein, in order to provide precise diagnoses and discover possible avenues for treatment. It is interdisciplinary, including infectious disease, oncology, inherited genetic disease, and legal issues such as parentage determination or forensic identity testing. While a variety of biophysical and biochemical techniques can be applied to study the molecular basis of disease, antibodies and nucleic acid probes are two of the principal approaches.
When monoclonal antibodies are either tagged to permit their detection or immobilized on a chromatographic column to purify their specific target molecule, they serve as powerful tools for analyzing pathologic processes. A monoclonal antibody conjugated to an enzyme generating a colored reaction product is the basis of the enzyme-linked immunosorbent assay (ELISA), which is widely applied in many diagnostic tests. Autoantibodies from the sera of individuals with autoimmune diseases are often used as highly specific reagents for understanding the nature of these diseases and the role that the affected molecules or organelles normally perform in the cell. These autoantibodies can be detected in tissues by using fluorescently tagged antibodies directed against human immunoglobulins.
In an analogous manner to immunohistochemistry, traditional histopathology can be enhanced by using in situ hybridization. With this technique, an infectious agent such as a virus or a specific messenger ribonucleic acid (mRNA) can be localized within a specific cell or tissue.
Diseases often result from germline or somatic mutations in the individual's DNA, such as are seen in sickle cell disease or cancer, respectively. These abnormalities can be detected by using two basic techniques of molecular genetics: the Southern blot and the polymerase chain reaction (PCR). For the Southern blot, high-molecular-weight DNA isolated from a specimen (most commonly peripheral-blood white cells) is digested by using an appropriate restriction endonuclease. The resulting fragments are then separated by gel electrophoresis and transferred to a nylon membrane, which is incubated with a solution containing a specific, labeled probe, also in single-stranded form. Probe-target hybrids formed by annealingof their complementary sequences can be detected by autoradiography or a colorimetric reaction. The Southern blot technique can detect DNA polymorphisms, mutations, or the presence of viral, bacterial, or specific sex chromosomes. In the polymerase chain reaction, short oligonucleotide primers flank the specific gene region or RNA sequence to be amplified and are combined with the target specimen and free nucleotides, which are synthesized into new DNA. An automated thermal cycler repeatedly alters the temperature to denature the target DNA, to allow the primers to reanneal to the target, and then to synthesize the product. This amplified polymerase chain reaction product is typically detectable as a band in a gel.
Understanding diseases at the genetic level has several advantages. Even when the gene's protein product is not expressed, definitive diagnoses can be made by using DNA-based techniques. Diseases that are similar clinically (phenotypically) can, in fact, be due to different mutations (genotypes) within a single gene or due to mutations in different genes, often related in an enzyme complex or as a portion of a group of structural proteins. Another advantage of molecular diagnosis is the ability to detect phenotypically normal carriers of genetic diseases in order to provide information for appropriate genetic counseling and prenatal diagnosis.