MEDICINE AND HEALTH/PHARMACEUTICAL PRODUCTS

Gene tests look for signs of a disease or disorder in DNA or RNA taken from a person's blood, other body fluids like saliva, or tissues. These tests can look for large changes, such as a gene that has a section missing or added, or small changes, such as a missing, added, or altered chemical base (subunit) within the DNA strand. Gene tests may also detect genes with too many copies, individual genes that are too active, genes that are turned off, or genes that are lost entirely.

Gene tests examine a person's DNA in a variety of ways. Some tests use DNA probes. A probe is a short string of DNA with base sequence complementary to (able to bind with) the sequence of an altered gene. These probes usually have fluorescent tags attached to them. During the test, a probe looks for its complement within a person's genome. If the altered gene is found, the complementary probe binds to it, and the fluorescent label can be used to identify the presence of the alteration.

Another type of gene test relies on DNA or RNA sequencing. This test directly compares the base-by-base sequence of DNA or RNA in a patient's sample to a normal version of the DNA or RNA sequence.

1. What methods based on DNA technology are used for detecting infectious diseases, such as AIDS, tuberculosis, Lyme disease, and human papilloma virus infection?

Direct detection of infectious agents from clinical specimens is based on three key principles: (1) all microbes contain a genome composed of DNA or RNA, (2) unique DNA or RNA sequences can be identified in each genome that are specific for the particular microbe, and (3) complementary primers to these specific sequences can be synthesized and used to bind to the target sequences present in the clinical specimen.

PCR technology facilitates the detection of DNA or RNA of pathogenic organisms and, as such, is the basis for a broad range of clinical diagnostic tests for various infectious agents, including viruses and bacteria. Scientists have also developed PCR-based tests designed to quantify the amount of virus in a person's blood ('viral load') thereby allowing physicians to monitor their patients' disease progression and response to therapy.

2. How is DNA analysis used to detect genetic diseases, such as cystic fibrosis, Duchenne’s muscular dystrophy, and Huntington’s disease?

PCR technology can be used to easily distinguish among the tiny variations in DNA that make people genetically unique.

There are two classes of genetic screening- i) presymptomatic screening -this is used to test individuals whose health is in danger ii) carrier screening - carried out in healthy individuals where genes are harmful to the health of their future offspring.Newborn screening is concerned with the analysis of blood or tissue samples taken in early infancy in order to detect genetic diseases for which early intervention can avert serious health problems or death. This started in 1960 with the ability to test newborns for a rare metabolic disease, phenylketonuria (PKU). Two other examples of newborn screening are the testing of African - American infants for sickle cell anemia and Ashkenazic Jews for Tay-Sachs disease.

Carrier screening is related to the analysis of individuals with a gene or a chromosome abnormality that may cause problems either for offspring or the person screened. This can be done by testing of blood or tissue samples and can indicate the existence of a particular genetic trait, changes in chromosomes, or changes in DNA that are associated with inherited diseases in asymptomatic individuals. Examples of carrier screening include sickle cell anemia, Tay-Sachs disease, Duchenne muscular dystrophy, hemophilia, Huntington's disease, and neurofibromatosis.

It should be noted that genetic disorders could be due to either a single gene disorder (disorder resulting from mutation affecting individual genes on a chromosome) or abnormal number or structure of chromosome. In single gene disorders, such as cystic fibrosis, the actual genes of the sampled embryo can be examined for the presence of the condition. Other genetic disorders, such as Duchenne's muscular dystrophy, or hemophilia, affect only males (sex linked diseases). In these cases, the cell is examined to determine the sex of the embryo and only female embryos are replaced.

In cases of recurrent chromosomal abnormalities such as Down's syndrome and recurrent miscarriages caused by parental translocations, the number and character of several chromosomes can be determined.

3. How can certain forms of cancer and diabetes be identified through DNA technology by genetic analysis? What is the mechanism by which gene therapy is used to treat certain forms of cancer?

Tests are targeted to healthy (presymptomatic) people who are identified as being at high risk because of a strong family medical history for the disorder. The tests give only a probability for developing the disorder. One of the most serious limitations of these susceptibility tests is the difficulty in interpreting a positive result because some people who carry a disease-associated mutation never develop the disease. Scientists believe that these mutations may work together with other, unknown mutations or with environmental factors to cause disease.

By taking a small genetic sample and copying it via recombinant cloning, a larger sample of the genetic material can be used to identify genetic mutations and abnormalities. The presence of breast cancer and neurofibromatosis may be indicated by the presence of mutated proteins in samples produced by recombinant cloning.

In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene. A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to take advantage of this capability and manipulate the virus genome to remove disease-causing genes and insert therapeutic genes.

Target cells such as the patient's liver or lung cells are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state.

4. How does deficiency in a certain protein contribute to ill health? How is DNA technology adapted to the biochemistry of these proteins?

Protein is necessary for the body to synthesize 13 amino acids and to break down polypeptidemolecules into the nine essential amino acids that the body cannot manufacture on its own. Collectively, these acids constantly work to replenish tissue in the body, so they play an important role in the maintenance of healthy bones, muscles, and organs. The body also usesprotein to produce hemoglobin in red blood cells, the vehicle by which oxygen is transported to muscles and organs. In addition, without sufficient protein, the lungs and immune system would cease to function properly.

Being able to express a recombinant human protein has revolutionized biomedical research. When a scientist has cloned a gene, he or she can compare it to a huge database of known gene sequences. If the gene has a sequence that is highly similar to a sequence of a gene of known function, he or she can predict the function of that gene. That knowledge suggests which experiments to perform with the product of the gene, which is frequently a protein.

If a scientist does know the function of a protein, overexpression can provide large quantities of the protein to study its biochemical properties. He or she can make targeted mutations and see what effects they have on the properties of the protein. Another reason to obtain large quantities of protein is to crystallize the protein and study its three-dimensional structure.

Nanotechnology + gene therapy yields treatment to torpedo cancer. March, 2009.

Results of world's first gene therapy for inherited blindness show sight improvement. 28 April 2008.

RNA interference or gene silencing may be a new way to treat Huntington's.

Sickle cell is successfully treated in mice. 

A combination of two tumor suppressing genes delivered in lipid-based nanoparticles drastically reduces the number and size of human lung cancer tumors in mice .

5. What are examples of human protein replacements? What therapies for genetic diseases are now available?

Recombinant human protein is human protein that is produced from cloned DNA. 

An example of a recombinant human protein that has no other source is the anti-anemia drug called erythropoietin. This hormone controls the production of red blood cells. It is used totreat anemia from various sources, including chronic kidney disease and cancer. Erythropoietin has also been used as a performance enhancement drug by athletes.

6. What are some biochemical problems encountered in producing pharmaceutical products by DNA technology? How can these dilemmas be circumvented?

Short-lived nature of gene therapy - Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.

Immune response - Anytime a foreign object is introduced into human tissues, the immune system is designed to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk. Furthermore, the immune system's enhanced response to invaders it has seen before makes it difficult for gene therapy to be repeated in patients.

Problems with viral vectors - Viruses, while the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient --toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.

Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy.

Source: http://molecular.roche.com/About/pcr/Pages/ApplicationsofPCR.aspx

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