Summary
Highlights
PCR (Polymerase Chain Reaction) is a powerful lab technique used to copy or amplify DNA. It requires DNA polymerase, primers, dNTPs (building blocks), a buffer, and the DNA to be copied. The process involves three main steps: denaturation (separating DNA strands by heating to 90°C), annealing (primers attach to target DNA at 40-65°C), and extension (DNA polymerase synthesizes new strands at 70°C). Each cycle duplicates the DNA, and special Taq polymerase (heat-stable) and thermal cyclers are used for rapid temperature changes.
qPCR allows real-time monitoring of the amplified DNA product. It uses a thermal cycler with a laser that illuminates reaction tubes. The DNA emits fluorescent light, captured by a detector, with higher fluorescence indicating more DNA. Two methods enable this: DNA-binding dyes like SYBR Green® that bind to double-stranded DNA after extension, and dye-containing probes (e.g., TaqMan® probe) with a reporter and quencher. The quencher blocks the reporter until the probe is cleaved by DNA polymerase during extension, allowing the reporter to emit fluorescent light.
RT-PCR allows RNA to be used as a template. Reverse transcriptase, an enzyme found in retroviruses, converts RNA into complementary DNA (cDNA). This cDNA is then amplified using the standard PCR method. The process involves a primer binding to RNA, reverse transcriptase synthesizing a cDNA strand, RNAse H digesting the original RNA, and DNA polymerase synthesizing a second cDNA chain. RT-PCR is commonly used for diagnosing diseases caused by RNA viruses, such as HIV.
Nested PCR addresses the issue of non-specific PCR products by using two sets of primers in two successive PCR reactions. The first reaction uses external primers that bind outside the target sequence, producing a slightly larger product that might include non-specific DNA. The second reaction then uses internal primers that are specific to the desired DNA sequence, significantly increasing the specificity of the amplification and eliminating non-specific products from the first round.
Touchdown PCR also aims to eliminate unwanted PCR products by modifying the annealing temperature. In the initial cycles, the annealing temperature is set higher than usual, making primer binding more stringent and favoring specific annealing. As cycles progress, the temperature is gradually decreased, which increases the reaction efficiency to produce a higher yield of the target sequence, building upon the specific products already generated at higher temperatures.
Hot Start PCR prevents non-specific amplification that can occur during the PCR setup at room temperature. It uses modified DNA polymerases that are inactive or blocked at room temperature. The polymerase only becomes active after the initial heating step (denaturation) in the thermal cycler. This ensures that primers bind specifically at higher annealing temperatures, reducing the formation of non-specific products.
Colony PCR is a technique used in bacterial transformation to identify colonies that have successfully taken up a desired DNA plasmid. Instead of extracting DNA, a portion of bacterial colonies is directly added to the PCR reaction mix. The initial heating step lyses the bacteria and releases the plasmid DNA. After PCR, gel electrophoresis is used to analyze the amplified products, indicating which colonies contain the target vector.