Applications Of Molecular Biology In The Clinical Laboratory
Diagnostic test based on molecular biology is rapidly becoming a very common procedure. In fact, molecular biology is considered as the technique of the future. Nowadays, molecular diagnosis is applied to various clinical conditions, including diagnosis of infectious diseases; viral infections, bacterial diagnosis, and genotyping; cancer diagnosis; mutation analysis; pharmacogenomics; chromosomal analysis for cytogenetic studies; and gene expression patterns in different disease states.
Polymerase chain reaction (PCR)
PCR is an in vitro DNA amplification procedure in which millions of copies of a particular sequence of DNA can be produced within a few hours. It is like a Xerox machine for gene copying. The reaction cycle has the following steps: (a) DNA strands are separated. (b) Then the primers are hybridized with their complementary DNA. (c) New DNA strands are then synthesized by the polymerase. Then the cycle (steps a, b, and c) is repeated. In each cycle, the DNA strands are doubled. By means of real time PCR, quantitation of the number of viruses present in a sample can be calculated, for example, viral load in HIV or HBV. So, the treatment modalities can be planned and the response to treatment can be assessed. In multiplex PCR, multiple primers are used to target multiple genes at once. Thus, a lot of information may be elicited from a single test run, which otherwise would require several reactions.
The reverse transcriptase PCR is used to convert the RNA to DNA. In ordinary PCR, DNA is detected; that DNA could be from a living or non-living organism. But in reverse PCR, mRNA is detected; that means, it is derived from a living organism. By this method, the presence of HIV RNA in blood could be detected as early as 4 weeks after infection.
Clinical applications of PCR
Bacterial and viral infections could be detected quite early. In early phases of tuberculosis, the sputum may contain only very few tubercle bacilli, so that usual acid fast staining may be negative. But PCR can detect even one bacillus present in the specimen. Any other bacterial infection can also be detected similarly. This technique is widely used in the diagnosis of viral infections like Hepatitis C, Cytomegalo virus, and HIV. Another important application is the diagnosis of genetic disorders, such as sickle cell anemia, beta thalassemia, and cystic fibrosis. PCR is especially useful for prenatal diagnosis of inherited diseases. PCR is also widely used for identification of mutations in cancer genes which will help to identify individuals having higher risk of cancer.
Microarray technique
A microarray is a series of thousands of microscopic spots of DNA probes on a slide, which can be used to analyze human samples. The DNA from the clinical sample (or PCR amplified DNA) is tagged with a fluorescent dye. These tagged DNA fragments are then incubated with the chip. After the hybridization is over, the microarray is scanned with a laser beam. Color intensity indicates the presence of active genes. If the patient has an undiagnosed fever, by doing the microarray test, the infectious agent (bacteria or virus) could be identified within a few hours. Upregulation of the genes in a cancer tissue could also be diagnosed easily.
DNA sequencing
This is the confirmatory test for mutations in genes. In automated instruments, Sanger’s technique is preferentially used. Nowadays, next-generation sequencers (NGS) are also becoming common in clinical laboratories, by which the specific gene could be sequenced within a few hours. Molecular biology offers highly sensitive yet specific diagnostic tests with quick turnaround time. In many cases, the diagnosis is based on detecting mutations in genes involved, but in some cases, it may be detecting changes in the gene expression also.
