Genetic testing

Gel electrophoresis

Genetic testing involves the direct examination of the DNA molecule itself. A scientist scans a patient’s DNA sample for mutated sequences.

There are two major types of gene tests. In the first type, a researcher may design short pieces of DNA (“probes”) whose sequences are complementary to the mutated sequences. These probes will seek their complement among the base pairs of an individual’s genome. If the mutated sequence is present in the patient’s genome, the probe will bind to it and flag the mutation. In the second type, a researcher may conduct the gene test by comparing the sequence of DNA bases in a patient’s gene to disease in healthy individuals or their progeny.

Genetic testing is now used for:

• Carrier screening, or the identification of unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to manifest;
• Confirmational diagnosis of symptomatic individuals;
• Determining sex;
• Forensic/identity testing;
• Newborn screening;
• Prenatal diagnostic screening;
• Presymptomatic testing for estimating the risk of developing adult-onset cancers;
• Presymptomatic testing for predicting adult-onset disorders.

Some genetic tests are already available, although most of them are used in developed countries. The tests currently available can detect mutations associated with rare genetic disorders like cystic fibrosis, sickle cell anemia, and Huntington’s disease. Recently, tests have been developed to detect mutation for a handful of more complex conditions such as breast, ovarian, and colon cancers. However, gene tests may not detect every mutation associated with a particular condition because many are as yet undiscovered, and the ones they do detect may present different risks to different people and populations.

Controversial questions

The bacterium Escherichia coli is routinely genetically engineered.

The absence of privacy and anti-discrimination legal protections in most countries can lead to discrimination in employment or insurance or other misuse of personal genetic information. This raises questions such as whether genetic privacy is different from medical privacy.

1. Reproductive issues. These include the use of genetic information in reproductive decision-making and the possibility of genetically altering reproductive cells that may be passed on to future generations. For example, germline therapy forever changes the genetic make-up of an individual’s descendants. Thus, any error in technology or judgment may have far-reaching consequences. Ethical issues like designer babies and human cloning have also given rise to controversies between and among scientists and bioethicists, especially in the light of past abuses with eugenics.
2. Clinical issues. These center on the capabilities and limitations of doctors and other health-service providers, people identified with genetic conditions, and the general public in dealing with genetic information.
3. Effects on social institutions. Genetic tests reveal information about individuals and their families. Thus, test results can affect the dynamics within social institutions, particularly the family.
4. Conceptual and philosophical implications regarding human responsibility, free will vis-à-vis genetic determinism, and the concepts of health and disease.

Gene therapy

Main article: Gene therapy

Gene therapy using an Adenovirus vector. A new gene is inserted into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a functional protein.

Gene therapy may be used for treating, or even curing, genetic and acquired diseases like cancer and AIDS by using normal genes to supplement or replace defective genes or to bolster a normal function such as immunity. It can be used to target somatic (i.e., body) or gametes (i.e., egg and sperm) cells. In somatic gene therapy, the genome of the recipient is changed, but this change is not passed along to the next generation. In contrast, in germline gene therapy, the egg and sperm cells of the parents are changed for the purpose of passing on the changes to their offspring.

There are basically two ways of implementing a gene therapy treatment:

1. Ex vivo, which means “outside the body” – Cells from the patient’s blood or bone marrow are removed and grown in the laboratory. They are then exposed to a virus carrying the desired gene. The virus enters the cells, and the desired gene becomes part of the DNA of the cells. The cells are allowed to grow in the laboratory before being returned to the patient by injection into a vein.
2. In vivo, which means “inside the body” – No cells are removed from the patient’s body. Instead, vectors are used to deliver the desired gene to cells in the patient’s body.

As of June 2001, more than 500 clinical gene-therapy trials involving about 3,500 patients have been identified worldwide. Around 78% of these are in the United States, with Europe having 18%. These trials focus on various types of cancer, although other multigenic diseases are being studied as well. Recently, two children born with severe combined immunodeficiency disorder (“SCID”) were reported to have been cured after being given genetically engineered cells.

Gene therapy faces many obstacles before it can become a practical approach for treating disease. At least four of these obstacles are as follows:

1. Gene delivery tools. Genes are inserted into the body using gene carriers called vectors. The most common vectors now are viruses, which have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists manipulate the genome of the virus by removing the disease-causing genes and inserting the therapeutic genes. However, while viruses are effective, they can introduce problems like toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, in order for gene therapy to provide permanent therapeutic effects, the introduced gene needs to be integrated within the host cell's genome. Some viral vectors effect this in a random fashion, which can introduce other problems such as disruption of an endogenous host gene.
2. High costs. Since gene therapy is relatively new and at an experimental stage, it is an expensive treatment to undertake. This explains why current studies are focused on illnesses commonly found in developed countries, where more people can afford to pay for treatment. It may take decades before developing countries can take advantage of this technology.
3. Limited knowledge of the functions of genes. Scientists currently know the functions of only a few genes. Hence, gene therapy can address only some genes that cause a particular disease. Worse, it is not known exactly whether genes have more than one function, which creates uncertainty as to whether replacing such genes is indeed desirable.
4. Multigene disorders and effect of environment. Most genetic disorders involve more than one gene. Moreover, most diseases involve the interaction of several genes and the environment. For example, many people with cancer not only inherit the disease gene for the disorder, but may have also failed to inherit specific tumor suppressor genes. Diet, exercise, smoking and other environmental factors may have also contributed to their disease.

Why does thymine replace uracil in DNA?

ONE 1

First, some clarification.As you already know, the difference between RNA (ribonucleic acids)and DNA (deoxyribonucleic acids) is the existence of a hydroxyl (-OH) groupon the 2' carbon of the ribose sugar in the backbone.
The removal of 2' hydroxyl groups from DNA does not occur afterthe DNA has been synthesized, but rather the 2' hydroxyl groups are removed from the nucleotidesbefore they are incorporated into the DNA.
During nucleotide synthesis, a portion of the nucleotide monophosphates (NMP's) are dehydroxylated to2'-deoxy-nucleotide monophosphates (dNMP's).
This means that GMP, AMP, CMP, and UMP are converted into dGMP, dAMP, dCMP, and dUMP, respectively.
However, before being incorporated into the chromosomes, another modification,using folic acid as a catalyst, methylates the uracil in dUMP to form a thymine making it dTMP.
After further phosphorylation, dGTP, dATP, dCTP, and dTTP can be used as the building blocks to construct DNA.
The important thing to notice is that while uracil exists as both uridine (U) and deoxy-uridine (dU),thymine only exists as deoxy-thymidine (dT).
So the question becomes: Why do cells go to the trouble of methylating uracil to thymine before it can be used in DNA?
The answer is: methylation protects the DNA.Beside using dT instead of dU, most organisms also use various enzymes to modify DNA after it has been synthesized.
Two such enzymes, dam and dcm methylate adenines and cytosines, respectively, along the entire DNA strand.
This methylation makes the DNA unrecognizable to many Nucleases (enzymes which break down DNA and RNA),so that it cannot be easily attacked by invaders, like viruses or certain bacteria.
Obviously, methylating the nucleotides before they are incorporated ensures that the entire strand of DNA is protected.
Thymine also protects the DNA in another way.
If you look at the components of nucleic acids, phosphates, sugars, and bases, you see that they are all very hydrophilic(water soluble).
Obviously, adding a hydrophobic (water insoluble) methyl group to part of the DNA is going to change the characteristics ofthe molecule.
The major effect is that the methyl group will be repelled by the rest of the DNA, moving it to a fixed position in the major groove ofthe helix.
This solves an important problem with uracil - though it prefers adenine, uracil can base-pair with almost any other base,including itself, depending on how it situates itself in the helix.
By tacking it down to a single conformation, the methyl group restricts uracil (thymine) to pairing only with adenine.
This greatly improves the efficiency of DNA replication, by reducing the rate of mismatches, and thus mutations.
To sum up: the replacement of thymine for uracil in DNA protects the DNA from attack and maintains the fidelity of DNA replication.(For another take on DNA, check out this article:Inhibition of Ribozymes by Deoxyribonucleotides and the Origin of DNA.)
[Moderator Note: In addtion, the cytosine base can spontaneously deaminate to form a uracil base, which would result inundetectable C -> U mutations if U were used routinely in DNA.
Since Thymine is basically methyl-U, the cell's DNA repair mechanisms can distinguish illegitimate U from legitimate methyl-U inDNA, and make the proper repair (replacing any U with a C).
C -> U mutations in RNA do not matter as much, because RNA is synthesized inlarge quantities and is rapidly degraded in comparison to DNA. -- Steve Mack, MadSci Moderator.]

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TWO 2

Why does RNA have uracil and DNA thymine?

Thymine in cells is made from Uracil in an energetically expensive
process, so we can assume uracil came first. Similarly sugars in
DNA (Deoxyribose) are made biosynthetically from those in RNA (ribose).
Cytosine degradation to form uracil is one of the most common
DNA mutations, but can be easily recognised and repaired.
If Uracil were present in DNA, the cell would not know which Uracil
bases to repair. Thus the use of thymine confers extra stability on
DNA. Stability that was not required in the more transient less
complex RNA world.

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THREE 3

How did the RNA world develop into the DNA world?

The million dollar question! Nobody really knows, many people have
suggested models all of which are difficult to prove.
->The first suggested stage is that RNA transferred most of its
catalytic functions to proteins, via an intermediate stage of
enzymes containing both RNA and proteins, of which a few remain
(i.e. ribosomes and telemorase). This is sometimes called the
ribonucleoprotein (RNP) world. DNA came later as its synthesis
requires several protein-only enzymes in all branches of life.
DNA provides much more chemical stability and double-strandedness
makes repair easier. RNA genomes would have had to have been
converted into DNA genomes. DNA can still be made from RNA
today by the enzyme reverse transcriptase found in many viruses.