Using the appropriate polymerase in PCR experiments will ensure the best yield and specificity, but there are so many polymerases on the market, how to choose, sometimes it is really a problem. Manufacturers will generally provide a selection guide listing the specificity, fidelity, yield, etc. of each product. Fidelity is one of the most critical factors for the success of the experiment and must not be overlooked.
What is polymerase fidelity?
The fidelity of DNA polymerase refers to the result of accurately copying the target template. Specifically, this involves multiple steps, including the ability to read the template strand, select the correct nucleoside triphosphate, and insert this nucleotide at the end of the 3 'primer. To efficiently distinguish between correct and wrong nucleotides, some DNA polymerases have 3 'to 5' exonuclease activity. This "correction" activity can excise the incorrectly incorporated single nucleotide and replace it with the correct nucleotide. High-fidelity PCR uses a DNA polymerase with low incorporation error rate and corrective activity to ensure faithful replication of the target DNA.
When does fidelity matter?
When designing PCR experiments, you must first consider whether your application requires high-fidelity polymerase. If the experimental results depend on the correct DNA sequence (such as cloning or sequencing), then you should try to minimize the incorporation of mismatched nucleotides. For ordinary PCR or colony PCR, to determine whether the amplicon is present or whether there is an insert on the plasmid, high fidelity is not necessary. Due to the powerful properties of certain high-fidelity polymerases, some researchers use them in all amplifications.
Manufacturers use various methods to determine the fidelity of DNA polymerases. Thomas Kunkel once introduced a method that uses part of the lacZα gene of phage M13 to check errors in DNA synthesis through changes in the color of host colonies. Based on Kunkel's analysis, Wayne Barnes developed an analysis that used PCR to replicate the entire lacZ gene and part of the two drug resistance genes, followed by ligation, cloning, transformation and blue-white screening. If there is an error in the lacZ gene, β-galactosidase activity is destroyed, resulting in the appearance of white spots. Through these experimental methods, the proportion of white spot clones can be converted into the amount of misincorporation. Sanger sequencing provides more direct reading of fidelity and can detect all mutations. Using this method, the entire mutation spectrum of the polymerase can be determined.
Regarding the high fidelity of DNA polymerase, there is no uniform definition. People often compare with Taq DNA polymerase to judge the relative fidelity. For example, scientists at NEB (New England Biolabs) have determined that the error rate of Taq polymerase is 2.7x10-4 ± 0.8x10-4, or 1 error per 3700 bases. These data indicate that using Taq to amplify a 400 bp fragment in 25 PCR cycles, half of the clones may have errors. For larger fragments such as 1 kb, each clone may carry unwanted mutations. In contrast, the error rate of NEB's Q5 super-fidelity polymerase is 100 times lower than that of Taq. Based on this data, 199 of the 200 clones will be correct.
Through the above introduction, I believe you have a certain understanding of the concept of fidelity. Before buying high-fidelity enzymes next time, in addition to looking at the introduction on the leaflet, you can also understand how they are measured so that they will not be blinded by various numbers.
Veneer molded doors with an antique finish refer to doors that are made using a combination of veneer and molding techniques to create a vintage or antique appearance. Let's break down the components:
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Veneer: Veneer is a thin layer of wood or wood-like material that is applied to the surface of a door to give it the appearance of solid wood. It is typically made from real wood veneers or engineered veneers, which are thin slices of wood or reconstituted wood fibers. Veneer provides a natural and authentic look to the door while being more cost-effective than solid wood.
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Molded: Molded doors are manufactured using a molding process that involves compressing wood fibers or engineered materials under heat and pressure. This technique allows for the creation of intricate designs, patterns, and contours on the door surface. Molded doors can imitate the appearance of panel doors or have raised or recessed designs.
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Antique Finish: The antique finish refers to a specific surface treatment or staining technique applied to the veneer or molded door to give it an aged or weathered look. This finish often involves distressing the wood, applying dark glazes or patinas, and adding subtle imperfections or wear marks. The goal is to create a vintage aesthetic that replicates the charm and character of antique doors.
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