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Applications of PCR


The Polymerase Chain Reaction (PCR) has found widespread application in many areas of genetic analysis. This is a list of some of these applications:

1 Medical applications
2 Infectious disease applications
3 Forensic applications
4 Research applications

Medical applications

PCR has been applied to a large number of medical procedures:

The first application of PCR was for genetic testing, where a sample of DNA is analyzed for the presence of genetic disease mutations. Prospective parents can be tested for being genetic carriers, or their children might be tested for actually being affected by a disease. DNA samples for Prenatal testing can be obtained by amniocentesis, chorionic villus sampling, or even by the analysis of rare fetal cells circulating in the mother’s bloodstream. PCR analysis is also essential to Preimplantation genetic diagnosis, where individual cells of a developing embryo are tested for mutations.

PCR can also be used as part of a sensitive test for tissue typing, vital to organ transplantation. As of 2008, there is even a proposal to replace the traditional antibody-based tests for blood type with PCR-based tests.
Many forms of cancer involve alterations to oncogenes. By using PCR-based tests to study these mutations, therapy regimens can sometimes be individually customized to a patient.

Infectious disease applications

Characterization and detection of infectious disease organisms have been revolutionized by PCR:

The Human Immunodeficiency Virus (or HIV), responsible for AIDS, is a difficult target to find and eradicate. The earliest tests for infection relied on the presence of antibodies to the virus circulating in the bloodstream. However, antibodies don’t appear until many weeks after infection, maternal antibodies mask the infection of a newborn, and therapeutic agents to fight the infection don’t affect the antibodies. PCR tests have been developed that can detect as little as one viral genome among the DNA of over 50,000 host cells . Infections can be detected earlier, donated blood can be screened directly for the virus, newborns can be immediately tested for infection, and the effects of antiviral treatments can be quantified.

Some disease organisms, such as that for Tuberculosis, are difficult to sample from patients and slow to be grown in the laboratory. PCR-based tests have allowed detection of small numbers of disease organisms (both live or dead), in convenient samples. Detailed genetic analysis can also be used to detect antibiotic resistance, allowing immediate and effective therapy. The effects of therapy can also be immediately evaluated.

The spread of a disease organism through populations of domestic or wild animals can be monitored by PCR testing. In many cases, the appearance of new virulent sub-types can be detected and monitored. The sub-types of an organism that were responsible for earlier epidemics can also be determined by PCR analysis.

Forensic applications

The development of PCR-based genetic (or DNA) fingerprinting protocols has seen widespread application in forensics:
In its most discriminating form, Genetic fingerprinting can uniquely discriminate any one person from the entire population of the world. Minute samples of DNA can be isolated from a crime scene, and compared to that from suspects, or from a DNA database of earlier evidence or convicts. Simpler versions of these tests are often used to rapidly rule out suspects during a criminal investigation. Evidence from decades-old crimes can be tested, confirming or exonerating the people originally convicted.

Less discriminating forms of DNA fingerprinting can help in Parental testing, where an individual is matched with their close relatives. DNA from unidentified human remains can be tested, and compared with that from possible parents, siblings, or children. Similar testing can be used to confirm the biological parents of an adopted (or kidnapped) child. The actual biological father of a newborn can also be confirmed (or ruled out).

Research applications

PCR has been applied to many areas of research in molecular genetics:

PCR allows rapid production of short pieces of DNA, even when nothing more than the sequence of the two primers is known. This ability of PCR augments many methods, such as generating hybridization probes for Southern or northern blot hybridization. PCR supplies these techniques with large amounts of pure DNA, sometimes as a single strand, enabling analysis even from very small amounts of starting material.

The task of DNA sequencing can also be assisted by PCR. Known segments of DNA can easily be produced from a patient with a genetic disease mutation. Modifications to the amplification technique can extract segments from a completely unknown genome, or can generate just a single strand of an area of interest.

PCR has numerous applications to the more traditional process of DNA cloning. It can extract segments for insertion into a vector from a larger genome, which may be only available in small quantities. Using a single set of ‘vector primers’, it can also analyze or extract fragments that have already been inserted into vectors. Some alterations to the PCR protocol can generate mutations (general or site-directed) of an inserted fragment.

Sequence-tagged sites is a process where PCR is used as an indicator that a particular segment of a genome is present in a particular clone. The Human Genome Project found this application vital to mapping the cosmid clones they were sequencing, and to coordinating the results from different laboratories.

An exciting application of PCR is the phylogenic analysis of DNA from ancient sources, such as that found in the recovered bones of Neanderthals, or from frozen tissues of Mammoths. In some cases the highly degraded DNA from these sources might be reassembled during the early stages of amplification.

A common application of PCR is the study of patterns of gene expression. Tissues (or even individual cells) can be analyzed at different stages to see which genes have become active, or which have been switched off. This application can also use Q-PCR to quantitate the actual levels of expression.

The ability of PCR to simultaneously amplify several loci from individual sperm has greatly enhanced the more traditional task of genetic mapping by studying chromosomal crossovers after meiosis. Rare crossover events between very close loci have been directly observed by analyzing thousands of individual sperms. Similarly, unusual deletions, insertions, translocations, or inversions can be analyzed, all without having to wait (or pay for) the long and laborious processes of fertilization, embryogenesis, etc.

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Non-invasive Prenatal Diagnosis using cell-free Fetal DNA

The holy grail of prenatal diagnosis has been the identification of chromosome and single gene abnormalities through maternal blood sampling. This would allow safe accurate prenatal diagnosis, requiring much lower operator skills, and automation is potentially possible, making it cost-effective. In contrast, standard prenatal screening involves ultrasound and biochemical risk assessment followed by invasive prenatal diagnosis by chorionic villus sampling (CVS), amniocentesis or fetal blood sampling – all requiring clinical expertise to perform and a variable risk of fetal loss.

The most common call is consistently from parents wrestling with the decision about whether to have an invasive test such as chorionic villus sampling (CVS) or amniocentesis. Because both procedures carry a one per cent risk of miscarriage, parents agonise over whether to put their pregnancy at risk in order to have conclusive information on a genetic condition. If cffDNA testing were to provide risk-free but reliable diagnoses of aneuploidies and other genetic conditions it would prove extremely popular with many parents.For most parents the earlier reassurance that cffDNA testing would bring will be welcome. However, we must avoid making assumptions that an earlier diagnosis will necessarily be easier for parents to cope with. Making painful decisions about the future of what is most often a wanted pregnancy is difficult at any gestation. Furthermore, there is no evidence that earlier terminations for fetal abnormality have substantially less emotional impact on women and couples than those carried out later in the pregnancy.

Another concern from the parental perspective is the form cffDNA testing takes, being a simple blood test. Women are very accustomed to having blood taken in pregnancy and while holding out an arm for a needle to be inserted is not always pleasant, it is something with which every pregnant woman is familiar. The ‘routine’ nature of the procedure could mean that some women embark on it without considering the possible implications and so are particularly distressed if test results bring unexpected news about the pregnancy. This has implications for how pre-test counselling and consent issues are handled.
In fact there is a precedent for a non-invasive diagnostic tool in routine use in antenatal care, namely ultrasound scanning. Every time an ultrasound probe is placed on the abdomen of a pregnant woman there is the possibility that an abnormality will be detected. Although information provision to women about the purpose of antenatal ultrasound is improving, we cannot underestimate the profound impact on parents when the scan shows that there is something wrong. However well-informed a woman may be, such news will always come as a shock and generate considerable anxiety and distress. This will also be the case for a diagnosis made from cffDNA testing, even if it comes earlier in pregnancy. 

For decades, attempts to identify intact fetal cells in the maternal circulation have been unsuccessful – too few cells were present, and the few that were identified could remain in the circulation for years. Cell-free DNA is also present in the circulation and probably arises from apoptosis (controlled cell death) of cells. Fetal DNA arises from dying trophoblast cells and comprises 3-6 per cent of the total cell free DNA in the maternal circulation. FfDNA was first demonstrated in the maternal circulation in 1997, it consists of short fragments of DNA, not whole chromosomes. It can be first identified from the fourth week of gestation and increases throughout pregnancy. It is rapidly cleared from the maternal circulation after delivery and is undetectable by two hours. DNA is normally transcribed in to RNA and cell free RNA (ffRNA) also circulates in the maternal circulation. It is more stable than other forms of RNA. Only genes that are being transcribed will produce RNA and therefore identification of free fetal against free maternal RNA may be possible through differential expression patterns (ie. different patterns of maternal and fetal gene activity).

Identification of the ffDNA from the free maternal DNA is a major challenge. Fifty per cent of the genes in fetal DNA will be the same as in the maternal DNA, as they originate from the mother. Two clinical uses of ffDNA technologies are currently used frequently; rhesus typing and fetal sexing. Rhesus blood grouping of the fetus in rhesus negative mothers is used to try and prevent isoimmunisation of the fetus by using anti D antibodies in mothers carrying rhesus positive babies. Fetal sexing is offered to women who are either carriers of an X-linked disorder and who only need to have a CVS if they are carrying an at risk male, or women at a one in four risk of having an affected baby with congenital adrenal hyperplasia. In these pregnancies early maternal treatment with dexamethasone hopefully prevents the development of the distressing problem of clitoromegaly in affected girls.

However, the above two uses for ffDNA are only the very beginning for what is potentially possible with this technology. Screening for Down syndrome and other major trisomies (conditions caused by having three, rather than two copies of a particular chromosome) would transform prenatal diagnosis and screening. Diagnosis could be earlier and available to a much wider number of women. Single gene disorders can also be potentially identified using ffDNA. At present this is being studied in cases where the father is a carrier of an autosomal dominant disease – identification of the mutation has to be from ffDNA as the mother does not carry the mutation. If the father carries a different mutation in an autosomal recessive disease then this might also be possible to identify. Another potential use for ffDNA is where ultrasound abnormalities are identified and it may be possible to confirm a diagnosis using genetic tests and ffDNA. Examples of this include achondroplasia and thanatophoric dysplasia. In view of the limited volume of ffDNA available, it is only possible to look at specific well recognised mutations rather than screening a whole gene. FfRNA from genes that are only active in the placenta will allow wider applications of the technology, as there will be different patterns of gene activity from maternal ffRNA, and hence differentiating between the two should be easier.

Greater availability of risk free tests for prenatal diagnosis may sound ideal, but first it is necessary to confirm the reliability and accuracy of the test. Fetal sexing had an approximately four per cent error rate until recently. Fetal sexing can be confirmed using ultrasound at 16 weeks so the error can be rectified, but for tests with no ultrasound markers this will not be possible. In addition, not all women want prenatal diagnosis but all women have blood samples taken during pregnancy, so they may not realise what is being tested for and receive results that they are ill-prepared to receive. Biochemical screening has had some similar problems but as it is a screening test the woman then has a choice whether to proceed to a diagnostic test. Lastly there is the possibility of abusing the technology particularly for fetal sexing; this has happened in other modalities of prenatal diagnosis in both ultrasound and karyotyping. There is a possibility of ffDNA testing being available over the internet and therefore more difficult to control.

FfDNA technology thus has the potential to change the face of prenatal diagnosis, allowing safer and earlier diagnosis for a wide number of genetic diseases, and we look forward to these advances with anticipation and a degree of impatience.

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ADVANCED MICROSCOPY TO SELECT BEST SPERM FOR IVF

Two independent studies presented at a European fertility meeting this week highlight new techniques for selecting those sperm most likely to result in successful pregnancies following IVF using intra-cytoplasmic sperm injection (ICSI). The first, by Italian scientists, uses high-powered microscopy to choose the healthiest-looking sperm for fertilisation, while the second, by US scientists, uses fluorescence microscopy to distinguish which ‘healthy-looking’ sperm are in fact harbouring DNA damage, which can decrease the chances of achieving pregnancy.
The Italian group, lead by Dr Monica Antinori, of RAPRUI clinic in Rome, used a microscope five times more powerful than standard microscopes to select sperm with the best genetic quality on the basis of their size and shape. When these sperm were each injected directly into an egg, they found that the overall pregnancy rate rose from 26.5 per cent in patients treated using standard methods, to 39.2 per cent in those treated with the new approach, known as intra-cytoplasmic morphologically-selected sperm injection (IMSI).
‘By treating this kind of patient with this technique, we offer them an opportunity to solve their fertility problems. As you can see from the results, the group that has had two or more IVF failures can get more than twice the opportunity to have a pregnancy with this new technique’, Dr Antinori  told the Times newspaper. But, being about twice as expensive as ICSI, which costs between £3,000 and £5,000 in the UK, the treatment does come at a price, warned Dr Antinori.
Meanwhile, the US group, led by Mr Conrado Avendano, of the Jones Institute for Reproductive Medicine in Virginia, found that 20-66 per cent of the sperm selected using conventional methods – which analyse size and shape using much lower power microscopy than the IMSI method – turned out to be harbouring DNA damage, suggesting that appearances can be deceptive and should therefore not be solely relied on.
‘DNA-damaged sperm has a highly deleterious effect on the ability to achieve a pregnancy. Even if damaged sperm are used and the woman becomes pregnant, the chances of miscarrying are significantly higher’, said Mr Avendano, speaking at the annual meeting of the European Society for Human Reproduction and Embryology (ESHRE).
While these studies have the potential to bring dramatic benefits to infertile males, they also highlight the need for further research to evaluate the various methods available for identifying the sperm most likely to result in pregnancy.

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Designer Babies Are Coming!

Designer babies with three parents could be born within three years. The controversial technique screens an embryo created by a man and a woman for incurable genetic diseases. Defective DNA is replaced with that from another woman, effectively giving the baby two mothers and a father.

Scientists at Newcastle University in the UK have already created embryos using the method and are perfecting it for use in IVF clinics.They say it could free children from diseases including some forms of diabetes, blindness and heart problems. Critics say it could lead to genetically-modified babies being designed to order. UK law says embryos created using the technique must be destroyed, but scientists hope this can be overturned.

The research focuses on mitochondria “batteries” inside cells, which turn food into energy. Each mitochondrion has its own DNA, which is passed from mother to child. Defects in this DNA affect more than one in 5000 babies and cause around 50 genetic diseases, some of which kill before adulthood. The researchers have managed to swap the damaged DNA with healthy genetic material.

The first step is fertilisation of an egg through IVF. The embryo is screened for defects. When it is a few hours old, the nucleus containing genetic information from the parents is removed and put into another woman’s healthy egg. Mitochondria are outside the nucleus so the baby is free of defects and will look like its “real” parents.

US biologist Professor Jonathan Van Blerkom says it would be “criminal” not to allow the technique to be used. There are fears that the influence of mitochondria on areas including longevity, IQ and fertility could lead to GM babies being made to order.

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