Quantitative analysis of specific mRNA transcripts using a competitive PCR assay with electrochemiluminescent detection
| Autor: | Ann Marie Szczepanik, Jeffrey A. Heroux |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Transcription
Genetic Pcr cloning Molecular Sequence Data Biology Polymerase Chain Reaction Sensitivity and Specificity Competitive pcr chemistry.chemical_compound Complementary DNA Genetics Animals RNA Messenger Genetics (clinical) DNA Primers Cerebral Cortex Messenger RNA Base Sequence RNA-Directed DNA Polymerase Molecular biology Rats chemistry Yield (chemistry) Luminescent Measurements Primer (molecular biology) Quantitative analysis (chemistry) DNA Spleen |
| Zdroj: | PCR methods and applications. 4(6) |
| ISSN: | 1054-9803 |
| Popis: | Reverse transcriptase-polymerase chain reaction (RT-PCR) has been widely utilized for both the qualita- tive and quantitative assessment of the levels of specific mRNA tran- scripts in living systems. Quantita- tion of specific transcripts has often proved to be problematic because of the difficulty associated with relating the PCR-amplified product to the starting cDNA representing the mRNA of interest. We have overcome these difficulties and have developed a competitive PCR assay employing the property of electrochemilumi- nescence for the detection of PCR products. This assay possesses the dual advantage of being both nonra- dioactive and highly sensitive. dividual PCR reactions can lead to signif- icant differences in product yield, thus confounding the interpretation of com- parative, quantitative measurements be- tween multiple samples; and (2) there is often a nonexponential accumulation of PCR product, commonly referred to as the "plateau effect," that typically oc- curs during the later cycles of PCR and is dependent on the starting template con- centration. As a result of these events, the amount of PCR product cannot reli- ably be related to the starting concentra- tion of target DNA and quantitative anal- ysis becomes impossible unless one determines PCR product yield over sev- eral cycles or template concentrations. These limitations have been overcome in large measure by the recent develop- ment of competitive PCR. (3A) In compet- itive PCR, a target sequence is coampli- fied in the presence of a competitor molecule that contains primer anneal- ing sites identical to that of the target molecule. However, the competitor mol- ecule differs in some other property, such as size or the presence or absence of a unique restriction enzyme site, so as to allow it to be distinguished from the am- plified target during the analysis of the PCR products. Because the ratio of target to competitor product is unaffected by the amplification efficiency during PCR, one is able to determine precisely the starting target template concentration by coamplifying varying amounts of competitor in the presence of a fixed concentration of target sequence and a limiting concentration of primers. We |
| Databáze: | OpenAIRE |
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