Using Genomic Mitochondrial Dna Medical Detection Disease Diagnosis Biology Essay


As medicine advances forward technologically, various tools have emerged to detect and diagnose diseases at earlier and earlier points in the progression of disease. Using cancer as an example to illustrate the progression of detection and diagnosis, one can see the improvement in medical technology. Detection of cancers has progressed from being found during post-mortems to being captured in imaging using techniques such as the use of magnetic resonance imaging (MRI)1. From imaging to capture existing cancers, further progress is being made by the use of PCR and associated genetic analysis techniques based on PCR2. PCR has been groundbreaking in that it analyses the genotype of cells and produces information on a molecular level, something that the imaging techniques cannot produce. Thus, this new information has shed light on another angle of disease, that of hereditary factors. A more recent innovation in the analysis of hereditary information is that of mini-sequencing. With the Human Genome Project providing the nucleotide sequence of the genome within a few years, identification of mutations that cause inherited diseases or predispose to acquiring diseases will become much easier. To do this successfully, efficient methods of analyzing individual sequence variation are required and mini-sequencing provides a solution to this.

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PCR is used to genotype single cells and the resulting information is valuable in detection and diagnosis. However, one of the problems encountered by many of the PCR techniques is that due to their high sensitivity, it is prone to a higher number of errors. Some of these errors include "potential sample contamination, total PCR failure, allelic drop‐out (ADO, when one of the alleles fails to amplify to detectable levels), and preferential amplification (PA) of one of the alleles"3. Therefore analysis of such results will run the risk of false positives in a high number of cases. False positives in the medical setting have greater consequences as it could lead to unneeded treatment and unnecessary trauma to the patient involved, if taken at face value. Therefore genetic analysis cannot rely on the use of PCR. A recent addition to the detection tool array is that of mini-sequencing. Often denoted solid- phase mini-sequencing, it is used for detecting single- nucleotide variations. This technique can be used to analyse both genomic DNA and mitochondrial DNA. In this technique, a detection primer is used to anneal to the nucleic acid target directly adjacent to a variable nucleotide position. A DNA polymerase is then used to specifically extend the 3' end of the primer with a radiolabeled nucleotide that is complementary to the variable site nucleotide. The figure below shows the steps of mini-sequencing4. With mini-sequencing in its present form, it can be used as a cost-effective screening process for both diseases that can be inherited and for factors that can predispose to the accruement of certain diseases.

One of the areas that immediately strike out as needing genetic analysis is that of foetal abnormalities. Called Preimplantation Genetic Diagnosis (PGD), it is currently used to help couples with a high risk of pregnancy with genetic abnormalities. PGD enables the genetic analysis for monogenic disorders of embryos that are generated by IVF techniques, thus allowing the implantation of embryos that have been unaffected. PGD uses the PCR technique5 but as discussed above, this technique is prone to errors. Various techniques have evolved, all based on PCR that are used successfully for mutation screening. These include techniques such as denaturing gradient gel electrophoresis6 and fluorescent PCR7. But as with PCR, the problems remain and often the results are time-consuming to obtain and require experience for data interpretation. In PGD, this is a problem as there are often only one or two blastomeres available to work from and the analysis has to be conducted with a day. Mini-sequencing overcomes these problems and allows the analysis and identification of specific mutations without having to sequence the entire product. Its efficiency and yield of good quality amplification products is an advantage also.

A study conducted by Fiorentino et al3 examined the use of mini-sequencing in PGD to detect and diagnose diseases such cystic fibrosis, sickle cell anaemia and retinoblastoma. In this study, PCR products from 55 PGD cases were simultaneously compared using traditional PGD techniques and mini-sequencing. They found that that mini-sequencing was accurate enough not only to detect mutations but also differentiate between the kinds of mutations. These results corresponded perfectly with the results obtained with sequence analysis. The study also found that the mini-sequencing method was very efficient in that it produced interpretable results for 96.5% of the blastomeres investigated compared to the 86.7% obtained by sequence analysis. A comparison of sensitivity showed that mini-sequencing is powerful enough to obtain informative results with as little as 1 nanogram of PCR product. As well as this, mini-sequencing involved computer-assisted analysis which allowed exact base identity and visualization of the mutation which eases data interpretation and reduces sources of error.

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The study conducted by Fiorentino et al not only showed how mini-sequencing can be used in PGD with efficient, accurate and sensitive results that are easy to interpret, they also emphasised the benefits of mini-sequencing as a medical technique. Each mutation investigated in this study was analysed with a common procedure for mini-sequencing. Thus the use of one procedure to detect a wide range of genotypes is undoubtedly an attractive one, in terms of efficiency and expense. That it is more sensitive and produces interpretable results on a more consistent level is an added bonus. Another useful feature of mini-sequencing is that different mutation sites can be analysed simultaneously, even when located in different regions of the gene. This reduces analysis time, something that is crucial especially in PGD but is also an attractive feature when detection and diagnosis is not set against time.

A study conducted by Poetsch et al8 used mini-sequencing to investigate the effects of nicotine exposure in Sudden Infant Death Syndrome (SIDS). Once PCR amplification had been carried out, the cases were analysed for twelve known polymorphisms using mini-sequencing. The study suggested that one of the major enzymes involved in the detoxification of nicotine, FMO3 has a G472A variant that is an additional genetic risk SIDS risk factor in children with smoking mothers. Such information has the potential of aiding the detection of infants with a higher risk SIDS and is the first step in attempting preventative measures for a disease that one is usually unaware of until it occurs. It also allows the narrowing of the disease epidemiologically, an invaluable process for the detection and diagnosis of disease, especially for screening. Hypothetically, this result could allow the screening of infants with smoking mothers to analyse their risk for SIDS, now that the demographic has been narrowed down (in an ideal world with no resource issues of course!). Also, with a legally heavyweight case such as that of SIDS, mini-sequencing allows objective evidence to be presented by the medical team.

One may wonder what the detection and diagnosis of a mutation can achieve apart from valuable information. A study conducted by Riccardi et al9 demonstrates the practical use of the information obtained from mini-sequencing. In this investigation, they looked into the CYP2D6 polymorphism, the gene which is involved in the metabolism of pesticides and drugs such as antidepressants, antipsychotics and antiarrhythmics. The polymorphism leads to variability in the metabolism of such drugs ranging from null to greater than normal activity. The study found that the African population studied had a greater frequency of greater than normal activity of metabolism of drugs. The implies that the genetic variation can account partially for adverse drug reactions, fatal drug poisoning and represent a risk factor for neurodegenerative diseases such as Parkinson's in people exposed to pesticides. The ramifications of this in personalised medicine are huge and can help physicians not only detect but predict the implications of their detection and treat accordingly.

With genomic analysis to detect and diagnose disease, one must not overlook the fact that mitochondrial DNA is also a culprit that could carry mutations that cause disease or cause predisposing factors that contribute to disease. An example of mitochondrial DNA carrying such mutations is that of the mitochondrial potassium channel, which has been investigated for its role in pulmonary hypertension10. If genomic DNA can undergo mini-sequencing to detect and diagnose disease, the same principles can be applied to mitochondrial DNA to detect and diagnose diseases that are caused by mitochondrial mutations. Whilst over 100 mutations are associated with human mitochondrial diseases11, only a small percentage of these mutations have been confirmed to be involved in diseases. The pathogenicity of a large number of these mutations rests on studies that are partially or completely flawed12. However, the small percentage of mitochondrial mutations that are associated with disease can be screened using mini-sequencing.

Using mini-sequencing to analyse mitochondrial DNA was initially used in forensic identification13. It allowed to individualise evidence in a courtroom setting and the technique's potential to analyse mitochondrial DNA was quickly realized. One study that investigated this potential is that of Álvarez-Iglesias et al14. This study focused on Leber disease which is a mitochondrially inherited degeneration of retinal ganglion disease15. In this investigation, they selected 25 mutations (21 confirmed in various studies as being pathogenic) and assayed them. They detected 11 mutations in 11 different patients suspected of a clinical diagnosis of Leber disease with no detection of false positives. They also found that there were advantages of using mini-sequencing compared to other techniques used for mitochondrial analysis in the clinical field. It allowed to genotype multi-locus mitochondrial DNA with low cost and high efficiency and the sensitivity was higher than sequencing16.

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Another study looked into the level of mitochondrial mutation. It must be noted at this point that genetic analysis can produce not only a discrete set of results (whether the mutation is present or not) but also a continuous set of results (the level of mutations present). Olsson et al17 investigated the level of mitochondrial mutation A3243G in individuals affected with mitochondrial diabetes and hearing loss (maternally inherited diabetes and deafness, MIDD). This mutation is carried by 80% of patients with MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes). The samples from PCR amplification were assayed using mini-sequencing and quantified. The results showed that the proportion of mitochondrial DNA with the A3243G mutation decreased over time and suggest that this may explain the observations of molecular genetic anticipation sometimes seen in mitochondrial disorders. This pattern of disease studied on a genetic level can help in the detection and diagnosis of diseases at various points in a patient's life so even at a point when the mutation levels are low, the sensitivity of mini-sequencing allows the mutation to be detected and the previous study of progress allows the diagnosis of the disease to be made.

With mini-sequencing used in both genomic and mitochondrial DNA analysis to detect and diagnose disease, the techniques in medicine have been pushed even further back in time in terms of diseases. The diseases now have the potential to be screened for and detected whilst in embryonic form (as shown by the use in PGD) thus the viability of embryos can be determined before implantation. Examples of such diseases that are detected include haemophilia and sickle cell anaemia, both diseases with very poor prognosis and a short life expectancy. Analyzing the factors involved can carry out detection and diagnosis of diseases whose onset isn't realized until they are fatal such as SIDS. In the example of SIDS, mini-sequencing has suggested that that an enzyme involved in detoxification of nicotine and thus the narrowing of the demographic for SIDS or pinpointing of the SIDS demographic can allow for more efficient screening. Mini-sequencing can also shed light on the future reactions of people to certain stimuli such as drugs. Investigations have shown that it can allow for treatment to be tailored according the polymorphisms detected thus producing optimal results. Mini-sequencing can also potentially warn physicians of diseases that can develop when a patient if exposed to a certain drug e.g. Parkinson's when exposed to pesticides and can therefore claim a preventative role in medicine.

Mitochondrial DNA is a slightly more controversial area to detect and diagnose disease using mini-sequencing. This is due to uncertainty surrounding the pathogenecity of many of the mutations in mitochondrial disease. However this is less to do with the technique of mini-sequencing and more to do with the actual reference points being disputed. In diseases such as Leber's where the pathogenecity of the mutation is not controversial, mini-sequencing can prove to help in detection and diagnosis. It can also help to assess the levels of mutation and thus map out the progress of disease. Indirectly, this is useful for detection and diagnosis as the progress of an individual can be assessed against the general progress, thus helping to arrive at an accurate prognosis.

Mini-sequencing has the potential to play a key role in the detection and diagnosis of disease in medicine, using both genomic and mitochondrial DNA. This does rest wholly on the mutations being mapped out in general before being applied to the individual. The sensitivity of the technique couple with the efficiency and cost-effectiveness make it an ideal contender for regular use in medicine when compared to the other variants of PCR techniques available. However, as with any technique used, caution must be exercised. Whilst the accuracy of mini-sequencing is higher than that of other techniques, it is not foolproof. False positives are still a risk, albeit a smaller one. One must bear in mind that it is a technique that could be used to help detect and diagnose disease and that means, it's a tool. The results produced must always be scrutinised with an open mind to avoid errors in interpretation, which can lead to errors in managing the disease.