General multiplex polymerase chain reaction realization method based on microarray chip

Abstract

The present invention provides a microarray chip-based multiplex PCR assay, comprising: (a) spotting primers into the hydrophilic microwells of the microarray chip; (b) covering the surface of the microarray chip with mineral oil; (c) performing the thermal cycling amplification; (d) using the amplified products as templates to conduct second round of PCR; and (e) reading the results of multiplex PCR reactions by electrophoresis or DNA-chip hybridization.

Description

technical field [0001] The invention relates to a method in the technical field of biomedicine, in particular to a method for realizing a general multiple polymerase chain reaction based on a microarray chip. Background technique [0002] Multiplex PCR is a special form of PCR (Polymerase Chain Reaction, polymerase chain reaction), which is characterized in that in the same reaction system, there are two or more pairs of primers for simultaneous amplification of different target fragments. Compared with conventional PCR, multiplex PCR has the advantages of synchronous detection of multiple targets, saving time, consuming less reagents, and requiring less sample volume. Therefore, it was first reported in 1988 (Chamberlain, J.S., et al., 1988. Nucleic Acids Res ., 16, 11141-11156), multiplex PCR has been successfully applied in many fields, including gene deletion analysis (Sieber, O.M., et al., 2002.Proc.Natl.Acad.Sci.U.S.A, 99,2954-2958 ), gene mutation and gene polymorphi...

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Problems solved by technology

Matsubara et al. (Matsubara, Y., et al., 2004. Anal. Chem., 76, 6434-6439) designed a chip for PCR amplification. The chip is a set of independent microwell arrays. Apply micro-spotting technology to add different samples (templates) and the same primers and other PCR reactions to each microwell, and then place the entire chip under the thermal cycle required for PCR to react. Although this method can achieve multiplex PCR And it needs a very small system to complete, but the addition of samples requires special instruments (microarray chip spotting system) and cannot be applied to various fields on a large scale

Ramalingam et al. (Ramalingam, N., et al., 2009. Biomed. Mibrodevices, 11, 1007-1020) integrated the idea of ​​a microfluidic chip, which is formed on a glass slide by combining structured PDMS with a slide Several reaction chambers, different primer pairs are added before the formation of the reaction chamber, while templates and other PCR reactants are added through microfluidic technology. Although this method can also achieve multiplex PCR, the maximum multiplex is low, and the chip cannot Repeated use, the whole process operation is very complicated

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