General multiplex polymerase chain reaction realization method based on microarray chip

Abstract

The invention relates to a general multiplex polymerase chain reaction realization method based on a microarray chip in the technical field of biomedicine. In the realization method, a microarray chip sample application system is used for respectively applying a plurality of pairs of primers to a plurality of hydrophilic micropores of the microarray chip, then a PCR (Polymerase Chain Reaction) component solution is added into the hydrophilic micropores in a pulling mode or a draining mode; the microarray chip of the microarray chip sample application system is placed on an in-situ PCR instrument or in a centrifugal tube filled with mineral oil for thermal cycle amplification, and finally the microarray chip is placed in the centrifugal tube or is directly used for centrifugally collecting PCR products to be used as a secondary amplification template; secondary amplification is carried out on the secondary amplification template by the plurality of primers for detection so as to realize the general multiplex polymerase chain reaction; and a result of the multiplex polymerase chain reaction is read by adopting agarose gel electrophoresis, polyacrylamide electrophoresis, capillary electrophoresis or a DNA chip.

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 synchronous 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 polymorphis...

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Problems solved by technology

Matsubara etc. (Matsubara, Y., et al., 2004.Anal.Chem., 76,6434-6439) have designed a kind of chip that is used for PCR amplification, and on the chip is a group of independent microwell arrays, application Micro-spotting technology adds different samples (templates) and the same primers and other PCR reactants to each microwell, and then the entire chip is placed under the thermal cycle required by PCR for reaction. Although this method can achieve multiple PCR and It can be completed with a very small system, 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 ​​microfluidic chips, forming several chips on a glass slide through the combination of structured PDMS and glass slides. Different primer pairs are added before the reaction chamber is formed, 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 be repeated. use, the whole process operation is very complicated

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