Polymerase Chain Reaction (PCR)
Conventional PCR Test Principle
Polymerase chain reaction (PCR) utilizes DNA polymerase, a naturally occurring enzyme that catalyzes the formation and repair of DNA 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 oligonucleotide primers. These primers are synthetic single-stranded DNA molecules that are complimentary to the ends of the targeted DNA region that determine the particular segment of DNA to be amplified. In each cycle, the solution is heated to separate the strands of target DNA and then cooled to allow the primers to base-pair to the complimentary target DNA sequence. At this point the DNA polymerase add nucleotides from the supply of dNTPs to the 3-prime end of each primer, thus, synthesizing a new DNA molecule that is complementary 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. The newly synthesized DNA fragments from the previous cycles serves as template for subsequent cycles. At the end of the thermocycling reaction, gel electrophoresis is then used to detect the presence of the target DNA. Gel electrophoresis is a technique that 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 the 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
Many of Viracor-IBT's infectious disease assays are designed on the principle of highly automated, real-time quantitative PCR. Unlike conventional PCR, real-time PCR allows simultaneous amplification and detection of the target. The accumulated PCR product is detected by monitoring increased fluorescence emission from the dye-labeled TaqMan® probes. The use of closed, optically clear tubes allows direct measurement of the fluorescent signal that is generated in solution. 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 of manual detection methods, such as gel electrophoresis.
The advantages of real-time PCR over conventional PCR include:
- Wide dynamic assay range, allowing maximum sensitivity
- Objective, quantitative results
- High degree of reproducibility
- Rapid turnaround time
- Minimized contamination risk
Real-Time PCR Test Principal
Viracor-IBT utilizes real-time PCR technology, which is based on detection of a fluorescent signal produced proportionally during the amplification of the target nucleic acid template. The patient sample is first processed to extract and purify nucleic acid (DNA or RNA). The purified nucleic acid 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 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 fluorescence detection allows acquisition of data when PCR amplification is still in the early exponential phase. Quantification of the target DNA or RNA template is determined by identifying the cycle number at which the reporter dye emission intensity rises above background noise. This cycle number is called the cycle threshold CT. The CT 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 CT 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-IBT. 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.
Gel Electrophoresis: http://www.dnalc.org/resources/animations/gelelectrophoresis.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#
Viracor-IBT PCR tests are performed pursuant to a license agreement with Roche Molecular Systems, Inc