Fornesic Medicine

The techniques of medical science applied to assist in the resolution of crimes, legal disputes etc. constitutes forensic medicine. Establishing the identity of victim (e.g of murder, accidents etc) is critical to solving the problems of crimes cases. DNA fingerprinting or DNA profiling, is a highly sensitive and extremely versatile approach to this problem.
Preparation of DNA sample: Common sources of DNA include blood, sermen solid tissures blood stains, semen stains, hair roots etc. The specimens should be stored under clean, dry, cool condition to minimize contamination by microorganisms and DNA degradation due to DNase .
The DNA is prepared as follows.
Cells lysed with the enzyme proteinase K which also digests protein residual protein may be separated using the detergent sodium dodecyl sulphate RNA can be eliminated by phenol pure high molecules weight DNA may be prepared using alternative protocol.
In case of mixed specimens, suitable separation procedures should be used in separate the constituent cells. For example viginal swabs from rape types can be extracted separately following specific procedures.
The degree of DNA degradation is assessed as follows soutnern blottin is combined with species specific probes to establish DNA identity of human origin or otherwise DNA degradation is estimated by electrophoresis in agarose gel staining with ethidium bromide and observation under UV light high molecular weight DNA appears as single large band whereas degraded DNA forms a large smear.
DNA quantity in the sample is determined by absorbance 260 nm agarose gel electrophoresis with DNA preparations of known quantity and staining with ethidium bromide to assess the intensity of stain taken and fixing the DNA on membrane filter and hybridizing with a labelled highly repetitive primate specific locus.
Approach of DNA analysis: Many polymorphic loci composed of tandem repeats of 2-80 base pair the number of repeats varying from 1-100 VNTR and polymorphic for the number of tandem repeats while minivariant repeats show polymorphism for base pair composition at specific sites within each repeat. This variation at one or more loci may be analyzed by RFLP or PCR can also be used to analyze polymorphic site that may or may not be located in tandem repeat units. The DNA sample or the PCR products may be analyzed by the following approaches.
VNTR analysis: The sample DNA is used for RFLP analysis this requires high molecular weight DNA PCR products are used to develop RAPD pattern this can use much smaller even somewhat degraded DNA preparations.
Dot blot Analysis: The DNA samples is subjected to PCR to amplify the loci polymorphism at which is be determined. The PCR products are fixed to membrane and hybridized with allele specific probes.
Base sequence Analysis: The base sequence of specific loci in the region of DNA can be determined variable region by DNA sequencing and used for preparing DNA profiles.

Gene Therapy

Human beings from more than 5000 different diseases caused by single gene mutations. e.g cystic fibrosis acatalasia Huntingtons chorea. Tay sach's disease Lesch-Nyhan syndrome sickle cell anemia, mitral stenosis, Hunter's syndrome haemophilia several forms of muscular dystrophy etc. In addition many common disorder like cancer, hypertension, atherosclerosis and mental illness seem to have genetic components.
Gene therapy may be defined in broad general terms as follows. Introduction of a normal functional gene into cells which contains the defective allele of concerned gene with the objective of correcting a genetic disorder or an acquired disorder. Application of gene therapy involves the following basic development in genetics molecular biology and biotechnology (1) determination of the role its products in health and disease (2) identification of the gene that play the key role in the development of genetic disorder (3) development of an approach for gene therapy.
The candidate disorder for gene therapy are selected on the basis of this following criteria (1) the disease should be life- threatening (2) gene responsible for the disease has been cloned (3) a precise regulation of the gene should not be required and (4) a suitable delivery system should be available.

Types of Gene Therapy

Gene therapy may be classified into the two types (1) germ line gene therapy and (2) somatic cell lgene therapy. In case of Germ line gene therapy germ cells sperms or eggs integrated into their genomes. Therefore, the change dur to which are ordinarily integrated into their genomes. Therefore, the change due to therapy is heritable and passed onto later generations. This approach theoretically is highly effective in counteracting the genetic disorders. However, this option is at least for the present for application in human beings for a variety of technical and ethical reasons.
In somatic cell gene therapy the gene is introduced only in somatic cells, especially of those tissues in which expression of the concerned gene is critical for health. Expression of the introduced gene relives symptoms of the disorder but this effect is not heritable as it does not involve the germline. Somatic cell therapy is the only feasible option, and clinical triasl have already started most for the treatment of cancer and blood disorders. This approach is divided into two categories.
(1) Augmentation therapy
(2) targetted gene transfer.

Augmentation Therapy

In this type of somatic cel gene therapy the functional gene introduced in addition so the defective gene endogenous to the cells the modified cell contain both the defective well as the normal copies of the gene. There are two general approaches to augmentation therapy. The first approach was used in the two general approaches to augmentation therapy. The first approach was used into the first two patients on whom gene therapy was attempted to correct the genetic disorder called Servere Combined Immune Deficiency syndrom produced by adenosine deaminase deficiency (1) normal gene copies was produced by cloning and the (2) packing into a defective retrovirus most of the viral genes were replaced by ADA gene(3) Lymphocytes were isolated from the patients and (4) the infected cells expressing the ADA gene were injected back the patients. The normal ADA gene was expressed the patients and ADA deficiency was portially corrected this resulted in an improvement in the patients immune system.
The variety of viral vectors have been used to deliver genes into the stem cells bone marrow cells cultured in vitro.
The second approach is the direct injection of DNA into the tissues either protein complexes into order to bring about receptor mediated transfer of DNA into specific tissue or even flaked DNA into muscle skin. Exciting result have been obtained with experimental hypercholesrolemia where IDI receptor levels have been augmented by injection of the gene as s sialoglycoprotein complex. THe problems in this approach as well related to the frequency of cells taking up and expressing the gene and more particularly the duration of expression. The gene delivery methods used for gene therapy can also be used for treatment of cancer. In case of cancer a toxin encoding gene can be delivered into the cancer cells. Similarly, appropriate interleukin genes can be delivered to boos the body defence mechanism.

Targetted Gene Transfer

Targetted gene transfer or gene targetting uses homologous recombination to repalce the endogenous gene with the functional introduced gene. The The first case of such gene transfer was used to disrupt the human B globin gene in cultured cells over through this approach more than 100 mammalian found through this sequence the site for recombination and is different from the gene to be introduced. Hence the sequence to be introduced is located in the inner region of the vector and is flanked by the sequences involved in recombination. A recombination of such a vector with its homologous cellular sequences produces duplication of the targetted sequences this is called insertional recombination. A strategy has been devised to modify only small sequence of the largest gene without the attendant gene duplication produced by insertional recombination. This approach, called in out method of gene targetting consists of the following two steps.
The first step called "in" steps, is targetted gene transfer using insertion vector the appropriately targetted cell will have gene duplication.
Second step termed as "out" step, depend on either intrachromosomal recombination or unequal sister chromatid exchange between homologous chromosome the recombination product of interest is chromosomes which has only a single and functional copy of the introduced gene.
The in-out strategy has been tested using the HGPRT gene. The gene was targetted into a mouse embryonic stem cell line subsequently it has been succsfully used with some other gene. This procedure is ideal for gene therapy.
Gene targettin is the strategy of choice for gene therapy for the following reasons.
(1) The targetted gene is changed in precise and specific manner.
(2) The introduced functional gene is placed in the same context it is flanked by the same DNA sequences as the replaced endogenous gene.
(3) No other gene of the genome is affected. The major limitation of the approach is the low frequency of homologous recombination this problem however is being removed by refinments of the teqchique. The feasibility of gene targetting has been demonstrated in number of different cell tyupes for several different genes. It is expected that targetted therapy would become a feasibility for many genes disease in the near future.

Drug Designing

This approach aims at designing drugs which specifically and selectively fit into the critical sites of the target molecules thereby inactivating the latter. The target molecule may be an enzyme concerned with either metabolism of DNA replication or hormone receptor or some other important molecule involved in a disease. A successful example of drug designing is provided by the drug proprandolol used for the treatment of heart attacks and hypertension was awarded J. Black the Nobel prize for physiology and Medicine in 1988 for this work Heart ailment are mainly due to an excess of the hormones norepineophrine and epinephrine, which act through two receptors called B propanolol blocks the B receptor thereby interfering with the action of these hormones.
Another drug called eimetidine blocks the H2 receptor in stomach lining of histamine histamine induces the release of HCl is stomach leading to the development of stomach ulcers. Cimetidine therefore, spacifically cures peptic ulcers by blocking the concerned receptor G Elison and G. Hitchings have used this approach to the useful drugs for the treatment of cancer gout, malaria and viral infections like herpes. Recently drug called azidohymidine has been developed for the treatment of AIDS this drug selectively inhibits the reverse transcriptase of HIV.
Another drug Ro-31-8959 designed to inhibit the HIV protease in stage 3 clinical trials using oral delivery inhibition of protease results in the production of immature non infections viral particles.

Disease Treatement

Treatment of diseases utilize a wide variety of preparations of both biological and abiological origins. The preparations of biological origin may either be crude ayuvedic medicines some allopathic drugs or purified to various degrees. Many of such compounds are obtained from plants but large number of them originate from micro organism cultured cells and recombinant organisms.
Micro-organism: A large number or pharmaceuticals originate from microorganisms they range from whole micro organism through biomass used as food supplements to a variety for highly valuable compounds like antibiotics, vitamins, enzymes, organic acids etc.
Plant Cell cultures: Some biochemicals of pharmaceutical value are produced by cultured plant cells e.g shikonin, berberine, ginseng biomass and taxol.
Animal cell culture: Cultured animal cells are the source of several compounds used in treatment of diseases e.g angiogenic factor, interlukin interferon.
Products from Recombinant Organisms: The product is obtained from nonrecombinant organisms are limited to their natureal capabilities. Genetic engineering has however, removed this limitation and genes from any organism can be transferred andexpressed into any other organisms.
Genetically Engineered Micro-organism: A large number of human genes encoding pharmaceutically valuable proteins have been cloned and expressed in micro-organisms. Initially E.coli was used as the host for obvious reasons of ease in cloning. But yeast is fast becoming the host of choice for production of recombinant proteins. Several of the recombinant proteins, used for treatment of diabetes dwarfism cancer interferons, interleukins granulocyte macrophage colony stimulating factors thromosis and AIDS.
Animal Cell Culture: More recently, cultured animal cells have been preferred for the expression of human genes encoding pharmaceutically valuable proteins. Some of these proteins have already been approved for therapeutic use tissue plasminogen activator erythroprotein and blood clotting factors the last two of the proteins have been approved for marketing in India.
Transgenic Plants: Plants are highly desirable in many of ways of commercial production of proteins of value. A large number of transgens encoding pharmaceutically valuable proteins have been expressed in plants. There is at least one example of commercial production of a recombinant proteins, hirudin, encoded by synthetic gene expressed in Brassica napus.
Advantage: Production or recombinant proteins of pharmaceutical value in microorganisms cultured animal cells or plants offers several important advantages over their conventional routes of productions.