Have you got the tips for multiplex PCR? |
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In the previous issue, we discussed the principles, advantages, and wide application of multiplex PCR technology. While the principle is identical to conventional PCR, practical implementation often presents challenges. Therefore, optimization is often necessary to achieve optimal amplification results. |
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Primer design In multiplex PCR, all primer pairs must be optimized to similar amplification conditions. Therefore, in addition to meeting general PCR primer design principles, primer design for multiplex PCR also requires attention to the following issues: primers must not be complementary to each other, especially at the 3' end to prevent dimer formation; primers must not be highly complementary to other amplified fragments or templates, and amplified fragments must not have significant homology; primer length (generally 18-24 nt), (G+C) content, and Tm value must be as consistent as possible; and the fragment sizes of the amplified products of each primer must vary to facilitate differentiation by electrophoresis. |
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Reaction system |
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The reaction system of multiplex PCR is one of the important factors for the amplification effect. Each component in the system must meet the amplification amount requirements of the target corresponding to each pair of primers. |
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Reaction conditions |
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Because a multiplex PCR reaction system involves multiple primer pairs and the template fragments being amplified vary in length, the amplification efficiency and speed of each primer pair also vary. Because multiplex PCR reactions always prioritize amplifying smaller fragments, the optimal PCR conditions required by each primer pair also vary. (When designing multiple primer pairs for multiplex PCR, the PCR amplification conditions required by each primer pair should be as consistent as possible.) Therefore, when selecting multiplex PCR amplification conditions (particularly annealing temperature and time), try to choose conditions that favor the amplification of larger fragments. |
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04 |
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Common Problems and Solutions |
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The main issues with multiplex PCR amplification include primer dimers, nonspecific amplification, and weak amplification product bands. In actual operation, we can make adjustments based on the following issues and their corresponding solutions. |
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Multiplex PCR requires specific amplification of multiple loci within a single reaction system. Therefore, primer pairing and competitive amplification can affect amplification performance. Choosing the right reaction system and conditions can improve multiplex PCR amplification performance. Therefore, in practice, experimental conditions should be optimized based on individual experimental circumstances to achieve optimal amplification results. |
