PCR, is the polymerase chain reaction, which refers to the addition of dNTP, Mg2+, elongation factors and amplification enhancement factors to the system under the catalysis of DNA polymerase, using the parent DNA as a template and specific primers as the starting point of extension , Through the steps of denaturation, annealing, extension, etc., the process of in vitro replicating daughter strand DNA complementary to the parent strand template DNA can quickly and specifically amplify any target DNA in vitro.
1. Hot Start PCR
The start time of amplification in conventional PCR is not to put the PCR machine into the PCR machine, and then the program starts to amplify. When the system configuration is completed, the amplification begins, which may cause non-specific amplification, and hot-start PCR can solve this problem.
What is hot start PCR? After the reaction system is prepared, the enzyme modifier is released at high temperature (usually higher than 90°C) during the initial heating stage of the reaction or “hot start” stage, so that the DNA polymerase is activated. The exact activation time and temperature depend on the nature of the DNA polymerase and the hot-start modifier. This method mainly uses modifiers such as antibodies, affinity ligands, or chemical modifiers to inhibit the activity of DNA polymerase. Since the activity of DNA polymerase is inhibited at room temperature, hot start technology provides great convenience for preparing multiple PCR reaction systems at room temperature without sacrificing the specificity of PCR reactions.
2. RT-PCR
RT-PCR (Reverse transcription PCR) is an experimental technique for reverse transcription from mRNA into cDNA and using it as a template for amplification. The experimental procedure is to extract total RNA in tissues or cells first, use Oligo (dT) as a primer, use reverse transcriptase to synthesize cDNA, and then use cDNA as a template for PCR amplification to obtain the target gene or detect gene expression.
3. Fluorescent quantitative PCR
Fluorescent quantitative PCR (Real-time Quantitative PCR, RT-qPCR) refers to the method of adding fluorescent groups to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and finally using the standard curve to quantitatively analyze the template. Commonly used qPCR methods include SYBR Green I and TaqMan.
4. Nested PCR
Nested PCR refers to the use of two sets of PCR primers for two rounds of PCR amplification, and the amplification product of the second round is the target gene fragment.
If a mismatch of the first pair of primers (outer primers) causes a non-specific product to be amplified, the possibility of the same non-specific region being recognized by the second pair of primers and continuing to amplify is very small, so the amplification by the second pair of primers , the specificity of PCR has been improved. One advantage of performing two rounds of PCR is that it helps to amplify sufficient product from limited starting DNA.
5. Touchdown PCR
Touchdown PCR is a method to improve the specificity of PCR reaction by adjusting the PCR cycle parameters.
In touchdown PCR, the annealing temperature for the first few cycles is set a few degrees above the maximum annealing temperature (Tm) of the primers. Higher annealing temperature can effectively reduce non-specific amplification, but at the same time, higher annealing temperature will aggravate the separation of primers and target sequences, resulting in reduced PCR yield. Therefore, in the first few cycles, the annealing temperature is usually set to decrease by 1°C per cycle to increase the content of the target gene in the system. When the annealing temperature is lowered to the optimum temperature, the annealing temperature is maintained for the remaining cycles.
6. Direct PCR
Direct PCR refers to the amplification of target DNA directly from the sample without the need for nucleic acid isolation and purification.
There are two types of direct PCR:
direct method: take a small amount of sample and add it directly to PCR Master Mix for PCR identification;
cracking method: after sampling the sample, add it to the lysate, lyse to release the genome, take a small amount of lysed supernatant and add it to PCR Master Mix, perform PCR identification. This approach simplifies the experimental workflow, reduces hands-on time, and avoids DNA loss during purification steps.
7. SOE PCR
Gene splicing by overlap extension PCR (SOE PCR) uses primers with complementary ends to make PCR products form overlapping chains, so that in the subsequent amplification reaction, through the extension of the overlapping chains, different sources of A technique in which amplified fragments are overlapped and spliced together. This technology currently has two main application directions: construction of fusion genes; gene site-directed mutation.
8. IPCR
Inverse PCR (IPCR) uses reverse complementary primers to amplify DNA fragments other than the two primers, and amplifies unknown sequences on both sides of a known DNA fragment.
IPCR was originally designed to determine the sequence of adjacent unknown regions, and is mostly used to study gene promoter sequences; oncogenic chromosomal rearrangements, such as gene fusion, translocation and transposition; and viral gene integration, are also commonly used now For site-directed mutagenesis, copy a plasmid with the desired mutation.
9. dPCR
Digital PCR (dPCR) is a technique for the absolute quantification of nucleic acid molecules.
There are currently three methods for the quantification of nucleic acid molecules. Photometry is based on the absorbance of nucleic acid molecules; real-time fluorescent quantitative PCR (Real Time PCR) is based on the Ct value, and the Ct value refers to the cycle number corresponding to the fluorescence value that can be detected; digital PCR is the latest Quantitative technology based on single-molecule PCR method for counting nucleic acid quantification is an absolute quantitative method.
Post time: Jun-13-2023