Novel Diagnostic approaches of Biotechnology
Disease diagnosis refers to identification of the cause of the disease. Conventional methods include microscopy, culture of specimen and testing for sensitiveness, several immunological assays etc. But these conventional mechanisms often have negative aspects like being tedious; taking longer time etc. In order to overcome this, various biotechnological approaches has been developed.
Small nucleotide sequences used in detection of complementary sequences in nucleic acid sample is known as probes. These probes can be radioactively or non radioactively labelled so that they can be used for detection purposes. The samples like blood fluids, tissues etc., can be analysed with probes for disease diagnosis. The main mechanism by which probes can be used is by:
(i)Hybridisation: The process by which DNA samples are allowed to bind to complementary probes for detection purposes is known as hybridisation. This may be by dot blot method, southern hybridisation. A probe hybridizes with a test sample only when the complementary sequences match. A sample preparation is done either on a solid support like nitro cellulose filter or it can be prepared in situ and used for in situ hybridisation. The probes which are used in diagnostic procedures are extremely sensitive to the causative organism. Hence a positive hybridization tests implies the presence of pathogen and thus the disease.
(ii) Ligase chain reaction: In the corresponding process, the DNA sample prepared is added to the reaction mixture having ligase enzyme and two oligonucleotide probes. The DNA sample pair with the complementary probes and a chain reaction is initiated. This when viewed under UV, bands corresponding to probes as well as those corresponding to target DNA is visible. The probes alone appear as a single band and the target DNA is flanked by two oligonucleotide probes and appear as a band which is equal in size to the sum of two probes.
The advantages of using a probe is that it is highly specific, and the procedure is relatively simpler and rapid. The results are obtained even when the amount of sample is less.
Monoclonal antibodies are a preparation of antibodies so that it is highly specific to a single epitope of an antigen. It is employed in immunological assays like ELISA, immuno PCR wherein monoclonal antibody specific for an antigen is attached with a marker and used for identification of specific antigen. This is also used in preparation of autoantibodies. Autoantibodies are produced by an organism in instances of auto immunity against its own organs. The antigenic specificities of such antibodies can be used in treatment of autoimmune disorders.
Detection of genetic diseases:
Genetic diseases are in born defects of a person. These are mostly caused due to single recessive mutation. Foetal cells are retrieved and diagnosed for any possible genetic diseases. The sample for such diagnosis is obtained from biopsies of trophoblastic villi which is an external part of human embryo. These can be used for detection of genetic diseases in many ways.
(i) Karyotyping of the cells helps in obtaining information about any chromosomal aberrations.
(ii) Assay of foetal cells reveals information about any defective enzymes produced relating to genetic diseases.
(iii) Modification of recognition site of restriction enzyme can occur as a result of genetic diseases. This can be detected by conducting RFLP analysis of foetal samples after southern hybridization process. For eg: in the case of sickle cell anemia, the defective and normal gene is analysed in this method revealing different band patterns in defective and normal individuals.
(iv) Oligonucleotide probes complimentary to mutated gene sequence caused as a result of genetic diseases and probes complementary to corresponding normal gene sequences are used to probe with the samples suspected of genetic diseases. The radioactive bands produced can be used to distinguish the former from the latter. A classic example is that of sickle cell anaemia. The sample is probed with probes complementary to the sequence altered by mutation and also with probes complementary to normal sequence. This is possible only in cases where, the bases of mutated sequence as a result of genetic disease as well as the normal unaltered sequence is known to allow synthesis of two probes for the technology.
These mechanisms have several advantages from conventional methods being comparatively powerful, and do not include risk of contamination as in the case of microbial culture method. It is even applicable in detection of pathogens which cannot be cultured. Another important application of these methods is that it has the capability to detect even latent viral infections.
Posted by: Alfia Sagheer
Department of Biotechnology