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How the Polymerase Chain Reaction is used in Medicinal Science and the Diagnosis of HIV

Introduction:

The Polymerase Chain Reaction (PCR) was discovered by Kary B. Mullis in 1984, for which he was awarded half the Nobel Prize for Chemistry in 1993. The polymerase chain reaction is a method of amplifying a sample of DNA exponentially in a shorter and simpler way than previous methods. Due to its simplicity the polymerase chain reaction has been adapted to significantly change the way certain medicinal and biological practices are carried out; "In terms of its power to drive biological research, the advent of the polymerase chain reaction can certainly be compared with the discovery of the techniques of molecular cloning some 20 years ago". In biology the process is used in the analysis of messenger RNA, as well as the amplification of DNA fragments for analysis. It has also been used for DNA sequencing, such as in the Human Genome Project. In medicine the polymerase chain reaction can be used to detect small amounts of nucleic acid, as in the diagnosis of disease. It can also be used for the detection of genetic mutation, new and known ones, such as the mutations that cause cystic fibrosis and muscular dystrophy.

The method of the polymerase chain reaction:

The polymerase chain reaction is a simple procedure that only requires 3 components. To start with a pair of primers are needed, one for each end of the target sequence. A primer is a short oligonucleotide that is complimentary to a sequence of DNA that flanks the DNA sequence that needs to be amplified. Also needed are all four of the deoxyribonucleoside triphosphates (dNTPs) that are the building blocks of DNA (see figure 1). Each dNTP contains the sugar deoxyribose, which forms the sugar backbone of the DNA molecule, a triphosphate group, and one of four bases: Adenine, Guanine, Cytosine and Thymine. These bases give the dNTP identity and are complimentary to each other: Adenine to Thymine and Cytosine to Guanine, which causes DNA to be double stranded (see figure 2).

A heat-stable DNA polymerase enzyme is the last component. This enzyme joins dNTPs together to form a complimentary strand to the template DNA. The polymerase chain reaction consists of cycles of the same three experimental steps (see figure 3). At first the sample DNA is heated to 95 oC. This melts the double stranded DNA helix into two separate strands. The DNA is then cooled to 54 oC; just cool enough to allow the primers to anneal to the beginning of the flanking DNA sequences on both strands. The mixture is then heated to 72 oC, which is the optimum temperature for the DNA polymerase enzyme. The enzyme then extends the primers using the four different dNTPs to form the complimentary sequence to the DNA, which can extend beyond the target sequence. The previous steps are then repeated in a new cycle, causing primers to anneal to the new single strands of DNA that were formed on the previous cycle. This creates new short single strands of DNA that include the primer sequences and the target region, as well as new long DNA single strands like the ones produced in the first cycle. A third cycle of the steps sees the production of double stranded versions of the short single strand created in the second cycle. This is the product wanted from the polymerase chain reaction. Ideally the amount of target DNA will increase exponentially according to the formula 2 n, where n is the number of cycles performed. After twenty cycles the original amount of DNA will be increased a million-fold.

The polymerase chain reaction can be used in its simplest form as just described in medicinal chemistry, but adaptations of this technique have been developed to specialise in certain areas. One of these adaptations is the quantitative polymerase chain reaction . This technique is used to amplify specific messenger RNA in a cell in order to have a large enough sample to physically measure. The messenger RNA will be amplified in the same reaction as a control sequence of known quantity in the cell. As both sequences will be amplified at the same rate the original amount of messenger RNA can be deduced through the comparison of the amplified messenger RNA against the amplified amount of the control (see figure 4). This technique is used to compare the levels of a specific messenger RNA in different cell populations, which can be used to discover whether a genetic mutation is being expressed in different tissues of the human body.

The application of the polymerase chain reaction as a HIV diagnostic tool:

The polymerase chain reaction is also used in the detection of HIV viral DNA. HIV is the Human Immunodeficiency Virus, which causes the destruction of the patient's immune system and can lead to the condition of AIDS. HIV has two main types: HIV-1 and HIV-2. These two types have different rates of transmission from person to person and also different prognoses of infection. This means that the different infections require different diagnosis techniques and treatments, in order to be effective. The polymerase chain reaction can be used to discriminate between the two types and whether a single or dual infection is evident. Antiretroviral treatment can slow the virus's progress in the body, and so early diagnosis of the disease can considerably increase the quality of life the patient has left. Antiretroviral treatment uses HIV antibodies to destroy the HIV DNA present in living cells, but the high rate of mutation in the HIV DNA means that it cannot be completely eradicated, making the disease incurable. The polymerase chain reaction can be used to detect mutations in the HIV DNA and help adjust treatment accordingly.

To diagnose HIV using the polymerase chain reaction, primers are made that are complimentary to a flanking sequence of a particular gene on the viral DNA. A sample is taken from the patient and all genetic material extracted. This genetic material is then subjected to the polymerase chain reaction using the specially made primers. If there is any HIV DNA present in the sample this will be amplified and available to analyse. This technique can also be used to detect HIV RNA.

Not only can the polymerase chain reaction be used to diagnose HIV in the first place, it can also be used to track the progress of patients, and determine how well they are responding to treatment. When patients are treated with antiretroviral drugs the quantity of HIV RNA can drop below detectable levels when using conventional methods. A quantitative method of the polymerase chain reaction can be used to measure the levels of HIV RNA in the patient. This measurement can then be used to determine the level of expression of the HIV virus in the body, as described earlier. This is an important marker in telling whether the patient is responding well to the drugs they are taking. Therefore the polymerase chain reaction can be a useful tool in making the treatment HIV patients receives more efficient.

As mentioned, the polymerase chain reaction can be used to detect mutations in the HIV viral DNA. This is important when trying to determine the source of therapy failure. A method of the polymerase chain reaction, called reverse transcriptase polymerase chain reaction (RT-PCR) is used to do this. RT-PCR involves taking messenger RNA from the infected cell and, using a reverse transcriptase enzyme and dNTPs, transcribing the single strand into double stranded circular DNA. This DNA won't be exactly representative of the template DNA that the messenger RNA was transcribed from, as all non-coding sections of the messenger RNA will have been removed before it is released into the cell. However, mutations in the non-coding DNA will not have any effect on any proteins produced and therefore are negligible. The transcribed circular DNA is then amplified using the traditional polymerase chain reaction technique, using DNA polymerase and the four dNTPs: dATP, dGTP, dCTP and dTTP. The amplified product is sequenced in order to detect any mutations along the target gene (see figure 5). Knowledge of which mutations have taken place is important in knowing which treatment to give the patient next.

The advantages of the polymerase chain reaction:

The advantages of using the polymerase chain reaction as a diagnostic tool for HIV are numerous. The polymerase chain reaction is known for its speed and simplicity. Thirty cycles of the polymerase chain reaction can increase the amount of the original DNA sample a billion-fold. This quantity of genetic material can be achieved in under hour. This is much faster than older, slower methods of DNA amplification, such as semi conservative replication. This means that results can be retrieved much more quickly, speeding up the diagnosis. Also the polymerase chain reaction requires only a small amount of genetic material in order to work. In theory only one strand of the target DNA is needed in order to amplify it, as long as it is complete and undamaged. Therefore, diagnosis can be made at far earlier stages of HIV infection than previously achieved, which can improve survival times. The simplistic and automatic nature of the polymerase chain reaction also means that it takes far less time to train people up in how to use the technique; "the complete beginner can start to perform polymerase chain reaction experiments and generate meaningful results within a few days at most". This means that the time taken and cost of training people to diagnose HIV can be significantly reduced. Another advantage is that the dNTP sequence of the target area of DNA does not need to be known in order to amplify that region. All that needs to be known is the sequences at either side of the gene as the positions of the primers makes sure that the DNA polymerase enzyme will automatically copy the area of interest. This means that mutations in the HIV genome will not effect its amplification and therefore the mutations can be copied and analysed.

The polymerase chain reaction is also extremely specific. It will only copy the target area and almost nowhere else along the DNA strand, due to the high levels of stringency at the high temperature of the reaction. Stringency is the how good the match is between the primer and its target. This can be controlled by temperature and salt levels. At high temperatures there is an extremely good match of primers to their targets, ensuring that they do not attach elsewhere on the DNA and cause non-target DNA to also be copied. Therefore, the results achieved through this technique will have a high level of accuracy. This is important in eliminating any false-positives or false-negatives in HIV diagnosis. Some conventional methods of diagnosis try to detect anti-HIV antibodies, which don't discriminate between different types of HIV leading to the wrong type of infection being diagnosed. In one study it was observed that many immunoblot assays, a conventional method, could produce false-positive results on dual infection with HIV-1 and HIV-2, when in fact only HIV-1 was prevalent. It was shown that a polymerase chain reaction method could prevent these false positives from occurring. The high specificity of the polymerase chain reaction detection of HIV-2 meant that it could be used as a standard to compare new more selective assays to.

The disadvantages of the polymerase chain reaction:

However, there are also disadvantages to the polymerase chain reaction. Although it has high specificity, this only works under certain conditions. The length of the target DNA sequence is a factor in the level of specificity. If the target sequence is too long then it is difficult to ensure that the target sequence will be copied in its entirety and accurately. Also if a primer sequence appears more than once in a DNA molecule then the primer may attach to more than one area, causing the experiment to copy more than one sequence or the wrong sequence completely. This can occur when certain subtypes of HIV-1 are present in the patient, causing false negative results. This is apparent in a documented case of an English tourist who contracted HIV while on holiday in Thailand. The patient displayed the symptoms associated with HIV and so was tested. HIV-1 proviral DNA PCR test where taken approximately 20 days and 26 days after infection and repeatedly gave negative results. Only 33 days after infection did the HIV-1 proviral DNA PCR test give a positive result. Due to this discovery there has been caution about using PCR for detection of HIV and it is recommended that any PCR results should be followed up by a conventional method of diagnosis.

Summary:

Overall it seems that the benefits of the polymerase chain reaction cannot be ignored. The faster and more accurate diagnosis of HIV patients is so important in ensuring that all patients are able to make the most of the time they have left. The polymerase chain reaction also has a use in tracking the progress of HIV patients. Only the polymerase chain reaction has the sensitivity to detect HIV RNA levels after antiretroviral treatment. Without this analysis tool the true state of HIV patients would not be known and their treatment could not be adjusted accordingly. Even though the specificity of the polymerase chain reaction is sensitive to its conditions, these conditions can be controlled so the optimum specificity can be achieved for every situation. The impressive speed, overwhelming simplicity, high sensitivity and great selectivity show that the polymerase chain reaction is too much of a useful tool in molecular medicine to dismiss over its few faults. It has developed so much in the past thirty years that it's impossible to predict what it will be used for in the next thirty, "The clinical usefulness of the polymerase chain reaction thus seems to be limited only by the power of our imagination in identifying specific targets."

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