Using the DNA from the parent strand as a template and specific primers as the starting point for extension is the fundamental idea behind polymerase chain reaction (PCR). The target fragment can be exponentially multiplied in a relatively short amount of time if the steps of denaturation, annealing, and extension are followed.
Different types of PCR are continually extended by continuous improvement and adjustment based on conventional PCR in order to meet the various requirements of actual experiments. These requirements include, but are not limited to, increasing specificity, reducing reaction time, amplifying complex templates, amplifying growing fragments, and other similar requirements.
According to the various techniques for carrying out the detection Endpoint PCR
Following completion of the PCR reaction, the final products of the PCR amplification reaction are subjected to qualitative analysis using various techniques such as gel electrophoresis, capillary electrophoresis, and so on. It is the oldest and simplest form of the freeze dryer technique, which is also the one that is used the most frequently these days. It is also referred to as conventional PCR.
Endpoint PCR is unable to monitor the progression of the reaction while it is occurring, and the only results that can be obtained are those obtained by electrophoresis after the reaction has been completed. These results, however, cannot be quantified on their own, and the quantity of product obtained does not necessarily reflect the initial situation.
Real-time PCR
The addition of fluorescent chemicals to the PCR reaction system is what is meant by the term "real-time fluorescence PCR." This allows the entire PCR process to be monitored in real time through the accumulation of fluorescence signals, and it ultimately leads to the achievement of quantitative analysis of the initial template through relative or absolute quantification. Both the fluorescent dye method and the fluorescent probe method can be used, but the distinction between the two is based on the fluorescent chemicals that are used.
Real-time fluorescent quantitative PCR can detect threshold fluorescence and is compatible with absolute and relative quantitative analysis. This means that the final result that can be obtained using this method can be either a specific copy number or a relative expression ratio.
Using digital PCR, an absolute quantitative analysis of the nucleic acid copy number can be accomplished by first dividing the reaction system equally into a large number of independent microreaction units for freeze dryer amplification, and then computing it using the Poisson distribution and the positive ratio.
The endpoint fluorescence can be detected with digital PCR, which also enables the calculation of specific copy numbers using absolute quantitative analysis.
Depending on the kind of sample being taken,
RNA is used as a template for reverse transcription PCR, cDNA is synthesized in the presence of reverse transcriptase, and then freeze dryer amplification is carried out with the cDNA itself serving as the template.
RT-PCR is frequently utilized for the diagnosis of genetic diseases as well as the analysis of gene expression in a wide variety of cell types, tissue types, and stages of development. The cDNA that is produced as a result of reverse transcription is put to use in a wide variety of experiments, some of which include the measurement of the levels of gene expression, the examination of the RNA virus content, the synthesis of cDNA probes, the cloning of genes, and the construction of cDNA libraries.
PCR 1 for the Colony
Picking the colony to be tested from the culture medium, mixing it into water, and then directly lysing it by either heating it or using a chemical reagent are the steps required to perform colony PCR, which is the PCR amplification of the exposed DNA as a template. The cultured bacterial broth is extracted directly, lysed in the same manner, and then the exposed DNA is used as a template for freeze dryer amplification in the process known as liquid PCR.
Colony PCR and liquid PCR are both the same in principle and processing; the only difference is that colony PCR includes a step in which individual colonies are screened to ensure that the template is free of trampy contamination. liquid PCR does not include this step.
PCR that is Direct 2
Direct PCR is the process of directly or simply lysing samples, which is then followed by PCR amplification without the need for an intermediate step of isolating and purifying nucleic acids. This cuts down significantly on the amount of time needed for the experiment and saves money.
During the phase of high-temperature denaturation, cells, tissues, and other materials are lysed in the buffer, which results in the release of DNA. Along with the DNA, cellular debris, proteins, lipids, and polysaccharides are also released, which has an inhibiting effect on the freeze dryer reaction. Therefore, for direct PCR, it is recommended to use DNA polymerases that have strong amplification properties, good resistance to inhibitors, and often higher sensitivity. This will allow for the successful amplification of trace amounts of DNA from unpurified samples.
Acres' ApexDirect DNA Polymerase (AG12205) is a high-performance DNA polymerase that has excellent amplification performance and broad template applicability for routine amplification
It is designated as product number AG12205
In addition to this, the product has a high level of resistance to inhibitors and is excellent for performing direct PCR reactions on samples such as blood and the tissues of animals and plants
It is able to effectively amplify for templates that have a high proportion of GC, a high proportion of AT, and a low starting amount
PCR with a significant amount of GC content3
- Because of the presence of three separate pairs of hydrogen bonds that are formed between bases G and C, the amount of energy necessary to unravel the chain is significant
- Due to the strong hydrogen bonds that exist between GC bases, it is challenging to open the template double strand during the amplification process when the template has a high GC content (G+C content of at least 60%)
- On the other hand, stable secondary structures are easily formed in templates with a high GC content, and these structures restrict the extension of DNA polymerase, which in turn has an effect on the synthesis of DNA
When amplifying fragments with a high GC content, it is difficult to denature the double-stranded template, and the primers are only weakly bound to the template. The most common strategy for overcoming the interference caused by the powerful hydrogen bonds that exist between G and C is to make use of PCR additives like DMSO or cosolvents that are designed to help denature DNA.
In the meantime, selecting a DNA polymerase with a high capacity for synthesis assists in the binding of primers to the template, which is necessary for the PCR amplification of templates with a high GC content. The use of a highly heat-stable DNA polymerase enables a higher denaturation temperature (for example, using 98 degrees Celsius as opposed to 94 degrees Celsius), which helps facilitate the dissociation of double strands of DNA and the PCR amplification process.
Conventional PCR reactions can amplify DNA fragments of up to 3-4 kb in size, whereas the term "long fragment PCR" refers to the amplification of DNA fragments that are larger than 5 kb.
Long fragment freeze dryer (Visit here) will typically use a combination of Taq DNA polymerase, which allows it to amplify long fragments in a short amount of time for high synthesis capacity, and high fidelity enzymes, which help ensure a low error rate for long fragment amplification. Taq DNA polymerase enables long fragment PCR to amplify long fragments in a short amount of time for high synthesis capacity.
Acres' L-ExpTaq DNA Polymerase is a high-fidelity enzyme that has been added to the superior Accurate Taq enzyme. This addition has given it partial 3′5′ Proofreading activity, which makes it ideal for PCR amplification of long fragments and more complex DNA.
The L-ExpTaq DNA Polymerase has been specially optimized to provide very strong amplification of long fragments, as well as the ability to amplify complex templates. This is one of the many purposes for which it has been developed. The L-ExpTaq DNA Polymerase is capable of amplifying up to 40 kb of DNA when using dsDNA as a template and up to 24 kb of DNA when using human genomic DNA as a template. It also has a high success rate for difficult-to-amplify templates that have a high percentage of GC in their sequences.
