Why pcr is important

Hodinka R. The clinical utility of viral quantitation using molecular methods. Huijsdens X. Quantification of bacteria adherent to gastrointestinal mucosa by real-time PCR. Igarashi T. Identification of mutans streptococcal species by the PCR products of the dex genes. Lett Appl. Rapid identification of mutans streptococcal species. Jordens J. Amplification with molecular beacon primers and reverse line blotting for the detection and typing of human papillomaviruses.

Kariyazono H. Rapid detection of the 22q Kearns A. Kim Y. The gene: the polymerase chain reaction and its clinical application.

Oral Maxillofac. Kimura S. Periodontopathic bacterial infection in childhood. Kostrikis L. Kubista M. The real-time polymerase chain reaction. Aspects Med. Kutyavin I. Nucleic Acids Res.

Diagnostic and prognostic relevance of expression of human telomerase subunits in oral cancer. Lehmann U. Detection of gene amplification in archival breast cancer specimens by laser-assisted microdissection and quantitative real-time polymerase chain reaction. Lyons S. Quantitative real-time PCR for Porphyromonas gingivalis and total bacteria.

Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating Epstein-Barr virus DNA. Mackay I. Real-time PCR in virology. Mauchline T. Quantification in soil and the rhizosphere of the nematophagous fungus Verticillium chamydosporium by competitive PCR and comparison with selective plating.

Matsuki T. Matto J. Detection of Porphyromonas gingivalis from saliva by PCR by using a simple sample-processing method. Maccartney H. Molecular diagnostics for fungal plant pathogens. Mitas M. Quantitative real-time RTPCR detection of breast cancer micrometastasis using a multigene marker panel. Morillo J. Quantitative real time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.

Morita E. Different frequencies of Streptococcus anginosus infection in oral cancer and esophageal cancer. Cancer Sci. Morrison T. Mullally B. Prevalence of periodontal pathogens in localized and generalized forms of early-onset periodontitis.

Mullis K. Target amplification for DNA analysis by the polymerase chain reaction. Murdoch-Kinch C. Oral medicine: advances in diagnostic procedures. Nakanishi H. Rapid quantitative detection of carcinoembryonic antigen-expressing free tumor cells in the peritoneal cavity of gastric-cancer patients with real-time RT-PCR on the lightcycler.

Nakano K. Demonstration of Streptococcus mutans with a cell wall polysaccharide specific to a new serotype K, in the human oral cavity. Nazarenko I. A closed tube format for amplification and detection of DNA based on energy transfer. Niesters H. Quantitation of viral load using real- time amplification techniques. Molecular and diagnostic clinical virology in real time. Novais C. PCR em tempo real. Nygren J. The interaction between the fluorescent dye thiazole orange and DNA. Okada M.

Detection of Actinobacillus actinomicetemcomitans and Porphyromonas gingivalis in dental plaque samples from children 2 to 12 years of age. PCR detection of Streptococcus mutans and S. Parra B. Detection of human viruses in periodontal pockets using polymerase chain reaction. Peter M. A multiplex real-time pcr assay for the detection of gene fusions observed in solid tumors. Quint W. Reliability of methods for hepatitis B virus DNA detection. Ranjard L. Characterization of bacterial and fungal soil communities by automated ribosomal intergenic spacer analysis fingerprints: Biological e Methodological variability.

Reis P. Quantitative real-time PCR identifies a critical region of deletion on 22q13 related to prognosis in oral cancer. Richard B. Identification of salivary Lactobacillus rhamnosus species by DNA profiling and a specific probe.

Riggio M. Ririe K. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Rio de Janeiro: Guanabara Koogan; Box Rupf S. Quantitative determination of Streptococcus mutans by using competitive polymerase chain reaction.

Oral Sci. Saarela M. Typing of mutans streptococci by arbitrarily primed polymerase chain reaction. Oral Biol. Sakamoto M.

Rapid deteccion and quantification of live periodontopathic bacteria by real time PCR. Detection of Treponema socranskii associated with human periodontitis by PCR. Santos C. Reverse transcription and polymerase chain reaction: principles and applications in dentistry. Saygun I. Detection of human viruses in patients with chronic periodontitis and the relationship between viruses and clinical parameters.

Schuurman R. Multicenter comparison of three commercial methods for quantification of human immunodeficiency virus type 1 RNA in plasma. Shelburne C. Quantitative reverse transcription polymerase chain reaction analysis of Porphyromonas gingivalis gene expression in vivo. Silva L. DNA Forense. Siqueira Jr. PCR methodology as a valuable tool for identification of endodontic pathogens.

Slots J. Rapid identification of important periodontal microorganisms by cultivation. Song Y. PCR-based diagnostics for anaerobic infections. Speers D. Diagnosis of malaria aided by polymerase chain reaction in two cases with low-level parasitaemia. Clinical applications of molecular biology for infectious diseases. Spolidorio D. Sterflinger K. A protocol for PCR in situ hybridization of hypomycetes. Takeuchi Y. One of the most remarkable and useful applications is the analysis of gene expression through the quantitative measurement of transcripts.

These standards can be internal or external. External standards may be homologous or heterologous. The standard is an RNA more rarely a DNA which is present in the RNA extract internal standard or which is added in known quantity in the reaction mixture external standard. The standard is amplified at the same time as the RNA of interest. There is therefore competition between the amplification of the standard and that of the DNA of interest.

The higher the standard quantity, the less the RNA of interest will be amplified and therefore its quantity will be small. Of course, the method of analysis of the PCR sample must make it possible to discriminate the standard with respect to the RNA of interest on the one hand and on the other hand to evaluate the relative amount of DNA of interest by comparison with the amount of standard that is known [ 48 ].

The internal standards are endogenous RNA, corresponding to RNA genes whose expression is presumed constant actin, beta2-microglobulin, etc. These standards have a major disadvantage: they require the use of primers different from those used for the RNA of interest. The kinetics of amplification are therefore substantially different, and it is very difficult or impossible to guarantee a constant expression between different samples.

The homologous external RNA standards are synthetic RNAs that share the same priming hybridization sites as the RNA of interest and that have the same overall sequence, with a slight mutation, deletion, or insertion that will allow the identification and quantification thereof with respect to the signal rendered by the RNA of interest.

These standards make it possible on the one hand to appreciate the variability introduced at the level of the RT and, on the other hand, generally have the same amplification efficiency as the RNA of interest whether it is at the RT level or PCR [ 48 , 49 ]. However, unlike homologous external standards, they have a different amplification efficiency compared to that of the RNA of interest. A dilution series is performed, each being used for amplification.

It is then a question of defining the ideal number of cycles to be placed in the exponential phase of the reaction while ensuring an effective amplification. Knowing the value of the signal measured on the sample to be quantified, the corresponding number of copies can be extrapolated from the curve. In the case of competitive PCR, a series of synthetic external homologous standard RNA dilutions are co-amplified with equivalent amounts of total RNA and thus an equivalent amount of the native gene [ 50 , 51 ].

The standard competes with the RNA of interest for polymerase and primers. As the standard concentration increases, the signal of the gene of interest decreases.

Here, the PCR does not need to be performed in the exponential phase and the results show a correct reproducibility. However, the method is cumbersome and does not allow to manage many samples simultaneously [ 52 ].

PCR is a fabulous diagnostic tool. It is already widely used in the detection of genetic diseases. The amplification of all or part of a gene responsible for a genetic disease makes it possible to reveal the deleterious mutations s , their positions, their sizes, and their natures.

It is thus possible to detect deletions, inversions, insertions, and even point mutations, either by direct analysis of PCR products by electrophoresis or by combining PCR with other techniques [ 53 ]. But PCR can still be used to detect infectious diseases viral, bacterial, parasitic, etc. Although other diagnostic tools are effective at detecting these diseases, PCR has the enormous advantage of producing very reliable and rapid results from minute biological samples in which the presence of the pathogen is not always detectable with other techniques [ 53 , 54 ].

In the context of genetic diseases, it is a question of detecting a mutation on the sequence of a gene. Several situations arise. The simplest ones concern insertions and deletions. In these cases, the mutation is manifested by the change in the size of the gene or part of the gene.

Insofar as the mutation is known and described, it suffices to amplify all or part of the gene. A deletion presents a contrary result [ 55 ]. The analysis of PCR products by electrophoresis, and therefore the evaluation of their size, leads directly to the diagnosis. The detection of inversions and point mutations is more delicate.

The difference in size between healthy and diseased DNA is zero in the case of an inversion and almost zero in the case of a point mutation. We cannot therefore retain the size criterion of the PCR products to achieve the result. It is therefore necessary to resort to techniques complementary to PCR.

Three approaches can be selected, the southern blot, the restriction fragment length polymorphism RFLP , or the detection of mismatch. The southern blot consists in hybridizing on the PCR product an oligonucleotide probe marked, thanks to a radioactive isotope or a fluorochrome, whose sequence is complementary and therefore specific to that which corresponds to the mutation. This strategy is well suited to inversion cases [ 56 , 57 ]. The RFLP can detect inversions such as point mutations.

It involves a restriction enzyme capable of hydrolyzing the PCR product at the sequence which sets the mutation. This approach is only possible if a restriction site is indeed present on this sequence, whether it is the mutated allele or the wild-type allele.

Mismatch detection is, like the RFLP, adapted to inversions and point mutations [ 57 , 58 , 59 ]. This mixture is then denatured by the temperature and then rehybridized. The mismatches concern a single base pair in the case of a point mutation and several base pairs in the case of an inversion.

These mismatches are then degraded by S1 nuclease, an enzyme that degrades only single-stranded DNAs. Another solution is to cleave the mismatches chemically osmium tetroxide, then piperidine , but it is more suitable for point mutations. In summary, mutation induces a mismatch at the level of enzymatic or chemical cleavage which leads to the generation of two fragments from a single PCR product. These fragments are analyzed by electrophoresis.

Contamination with viruses or microorganisms bacteria, parasites, etc. PCR is therefore a tool all the more effective in detecting the presence of a pathogen in a biological sample that its sensitivity and specificity are very large.

The performance of the PCR diagnosis is essentially based on a criterion: the choice of primers capable of very selectively amplifying a sequence of the DNA of the virus or microorganism [ 57 , 58 , 59 ]. Matrix DNA, on the other hand, must be extracted from a tissue in which the microorganism is present. It is therefore sufficient to amplify a specific sequence of the pathogen from a sample taken on the patient and to analyze the PCR product by electrophoresis. The size of the amplified DNA fragment, which must conform to the expected size, guarantees the reliability of the result and therefore of the diagnosis.

This method, quite reliable and inexpensive, nevertheless has some disadvantages. False positives are quite common because of cross-reactivities.

Positive samples are therefore tested for control by another routine technique, Western blot. The blood of these newborns usually contains anti-HIV antibodies of maternal origin and they are therefore seropositive. On the other hand, they do not necessarily carry the virus. In this type of case, the PCR diagnosis is relevant [ 57 , 58 , 59 , 60 ]. The method involves amplifying a specific sequence of the provirus from a lymphocyte extract. The same principle is used for the detection of toxoplasma in newborns whose mother is a carrier.

Quantitative or semi-quantitative methods have been developed which also make it possible to evaluate the viral load. PCR is remarkably effective at identifying species, varieties, or individuals by genetic fingerprinting. This application is based on the knowledge acquired on genome structure. It is simply to amplify nucleotide sequences that are specific to species, variety, or individual.

In eukaryotes, in particular, these sequences are very numerous and offer a vast palette that allows identification in a very precise and very selective way. Indeed, the genomes of eukaryotic organisms have, unlike prokaryotes, coding sequences and noncoding sequences. Infectious Disease Detection and Identification Detection of the Human Immunodeficiency Virus HIV , one of the most difficult viruses to detect, and other disease organisms such as those that cause middle ear infection, tuberculosis and Lyme disease.

Early detection of several forms of cancer including leukemia and lymphoma. Detection of viral DNA and virulent sub-types, including those that caused earlier epidemics. Production of hybridization probes for both northern and southern blot hybridization. Analysis of DNA from ancient sources. Want more Protein Man blogs? Features Sensitivity: Linear responses over the range of 0. PCR is a common tool used in medical and biological research labs.

It is used in the early stages of processing DNA for sequencing , for detecting the presence or absence of a gene to help identify pathogens during infection, and when generating forensic DNA profiles from tiny samples of DNA. How does PCR work? We will explain exactly what each of these do as we go along. PCR involves a process of heating and cooling called thermal cycling which is carried out by machine.

There are three main stages: Denaturing — when the double-stranded template DNA is heated to separate it into two single strands. Extending — when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme.

These three stages are repeated times, doubling the number of DNA copies each time. A complete PCR reaction can be performed in a few hours, or even less than an hour with certain high-speed machines.