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Conventional PCR Test Principle
Polymerase chain reaction (PCR) utilizes DNA polymerase, a naturally occurring enzyme that catalyzes the formation and repair of DNA and RNA in creating a chain reaction that produces unlimited DNA copies from a single molecule of genetic material. The targeted DNA is incubated in a test tube with heat-resistant DNA polymerase, a supply of nucleotides called dNTPs and primers. Primers are synthetic single-stranded DNA complimentary to the ends of the targeted DNA, therefore determining the particular segment of DNA to be amplified. In each cycle, the solution is heated to separate the strands of the target DNA and then cooled to allow the primers to hydrogen bond to the complimentary target DNA sequence, at which point the DNA polymerase adds nucleotides from the supply of dNTPs to the 3-prime end of each primer, thus synthesizing new DNA identical to the original starting target DNA. The first cycle produces two double-stranded DNA molecules identical to the starting target DNA material. The cycle is then repeated many times, each time producing double the amount of target DNA. Gel electrophoresis is then used to detect the presence of the target DNA. The technique separates nucleic acids or proteins based on their rate of movement through a gel in an electric field. Since DNA is negatively charged, the molecules migrate to the positive electrode. The distance a DNA molecule travels is inversely proportional to molecular size; longer molecules travel more slowly through the gel. When the current is turned off, the DNA molecules are viewed under ultraviolet light. The DNA strands are arrayed in bands along a lane; shorter molecules travel the farthest and are the bands at the bottom of the gel.
The Difference Between Conventional PCR and Real-time PCR
The majority of ViraCor’s assays are designed on the principle of highly automated, real-time quantitative PCR. Unlike conventional PCR, the real-time PCR technique allows the simultaneous amplification and detection of the target. The accumulated PCR product is detected by monitoring changes in fluorescence emission from the dye-labeled TaqMan® probes. The accumulated fluorescence PCR product is generated in optically clear PCR tubes. The use of closed, optically clear tubes allows fluorescent signal that is generated in solution, which can be measured directly. Since there is no need to open tubes for post-PCR handling steps, the risk of contamination is minimized. Real-time PCR also eliminates the subjectivity that plagues manual detection methods, such as gel electrophoresis.
The advantages of real-time PCR over conventional PCR include:
- Wide dynamic range of assay, allowing maximum sensitivity
- Objective, quantitative results
- High degree of reproducibility
- Rapid turnaround time
- Minimized contamination risk
Real-time PCR Test Principle
ViraCor utilizes real-time PCR technology, which is based on detection of a fluorescent signal produced proportionally during the amplification of a PCR product. The patient blood sample is processed to extract viral DNA from the plasma. The purified DNA is then added to a prepared PCR master mix for thermocycling. The master mix contains all the necessary ingredients to produce DNA through the polymerase chain reaction including MgSO4, polymerase, dNTPs, PCR buffer, and appropriate primers and probe. A probe is designed to anneal to the target sequence between the traditional forward and reverse primers. The amount of fluorescence released during the amplification cycle is proportional to the amount of the product generated in each cycle. The sensitivity of detection allows acquisition of data when PCR amplification is still in the exponential phase. This is determined by identifying the cycle number at which the reporter dye emission intensity rises above background noise. This cycle number is called the threshold cycle. The threshold cycle is determined at the most exponential phase of the reaction and is more reliable than end-point measurements of accumulated PCR products used by traditional PCR methods. The threshold cycle is inversely proportional to the copy number of the target template, as in the higher the template concentration, the lower the threshold cycle measured.
A screen snapshot of a typical TaqMan® ABI PRISM run at ViraCor. The lower the cycle number, the greater the quantity of target present. Therefore, curves furthest to the left have higher copy numbers; curves to the right of the screen represent lower copy numbers.
EXCELLENT TUTORIAL LINKS
PCR: http://www.dnalc.org/ddnalc/resources/pcr.html
Gel Electrophoresis: http://www.dnalc.org/ddnalc/resources/electrophoresis.html
Real-time PCR: http://pathmicro.med.sc.edu/pcr/realtime-home.htm
Conventional vs Real-time PCR: http://pathology2.jhu.edu/molec/techniques_main.cfm#
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