![]() If multiple peaks were detected, it would indicate that multiple PCR products were produced and the PCR assay would require further optimization and development. The presence of a single peak indicates the presence of a single amplicon at the end of the PCR reaction. See Figure 4 for an example of a melt curve of a single PCR product. The increased specificity is achieved through the use of specific NA probes and/or a melt curve analysis that follows the PCR reaction. Starting quantities of a sequence of interest can be determined by comparison of samples to a standard curve of known quantities of DNA. Further, real-time PCR can be both quantitative and specific. First, the PCR product can be detected in real time, so the need for an agarose gel to visualize the DNA post-PCR is unnecessary. Real-time PCR, also called qPCR (quantitative PCR), is a more recent but already extremely common method of PCR that offers several advantages over conventional PCR. Limitations of this type of end-point PCR include low sensitivity and non-quantitative results. Figure 2 gives an example of an agarose gel with a DNA ladder on the left and several PCR reactions to its right. The bands may be isolated from the gel, and the DNA can be purified and sequenced, which is a more dependable means of specificity determination than sizing via comparison to a DNA ladder. The specificity of the PCR reaction is confirmed by size as compared to a DNA ladder, which is a mixture of known sized fragments of DNA. The DNA is visualized by using an intercalating dye such as ethidium bromide or Sybr Safe, and a UV light source. Often, conventional PCR reactions are loaded onto an agarose gel and are resolved by size via electrophoresis. A major advantage to conventional PCR is the ready access to conventional thermocyclers that almost all research facilities have, and the fairly low cost. It gives qualitative results and requires a post-PCR step for detection or visualization of the DNA. Standard or conventional PCR is the most basic type of PCR reaction. This is the point at which the PCR reaction product is analyzed via gel electrophoresis for conventional PCR reactions.Ī wide variety of PCR methods exist, and each has advantages and limitations. The final stage is the plateau phase, which occurs when the reaction has stopped, and no additional amplicon is being generated. The linear phase occurs as the reaction is slowing due to the consumption of the reagents and the degradation of the products. Real-time PCR detection is carried out during this exponential phase. The exponential phase is the period in which exact doubling of a nucleic acid product occurs every cycle. The basic PCR reaction occurs in three phases. This leads to the exponential amplification of the original template, often resulting in millions or billions of copies of the original NA target. Each round of temperature cycling results in two times more target sequence than the prior round. For instance, the amplification of very short target sequences requires much shorter incubations at each temperature than that of very large target sequences. The time spent at each temperature can be optimized for specific assays. Conventional cycling conditions are 95✬ for 5 minutes initially to denature all template NA, followed by 2-40 repeats of 95✬ for 30 sec, 60✬ for 30s and 72✬ for 1 minute. Extension of the primers with nucleotides by the DNA Polymerase is achieved at temperatures ranging from 60-72✬. The temperature is cooled to 37-60✬ to anneal primers to the target sequence. Denaturation of the NA template sequence is achieved at 95✬. The PCR reaction mixture is temperature cycled, typically 20-40 times. This enzyme adds the deoxyribonucleoside triphosphates (dNTPs or nucleotides) onto the ends of the primers to extend the NA based on the template NA sequence. A variety of DNA Polymerases have been utilized for PCR, but the thermostable Taq DNA Polymerase is probably the most widely used. Primers ideally lack secondary structure and are not complementary to each other, to prevent primer-dimer formation. They are designed to anneal to opposite strands of a specific NA template target and usually are between 15-40 bases long. Primers are short sequences of nucleotides synthesized in vitro. The template for a PCR reaction may be any nucleic acid sequence of interest, and the NA source may be DNA, RNA, or cDNA. PCR reactions amplify target nucleic acid sequences via the use of a DNA Polymerase, primers, and nucleotides. ![]()
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