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In-situ polymerase chain reaction approach for detecting target gene on chip

A technology for detecting targets and genes, which is applied in the field of in situ polymerase chain reaction on the surface of gene chips to detect target genes, and can solve problems such as the need for pre-amplification, not a detection method, and difficulty in application and promotion.

Inactive Publication Date: 2006-12-20
SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI
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Problems solved by technology

Directly using clinical samples as templates, in the solid-phase PCR reaction, there is a problem that internal primers and external primers compete with each other, which affects the amplification efficiency of PCR; However, pre-amplification before sample hybridization is still required, so it is not a very ideal detection method
Moreover, there are very few commercially available slides modified by mercapto silanization, which brings difficulties to the application and promotion of this method.

Method used

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  • In-situ polymerase chain reaction approach for detecting target gene on chip
  • In-situ polymerase chain reaction approach for detecting target gene on chip

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Embodiment 1

[0017] Using the method provided by the invention, the detection of the commonly used plasmid carrier pCAMBIA1301 in transgenic plants is discussed, and the preliminary detection of 4 genes GUS, 35S promoter, hpt and aadA contained in the plasmid is made. The chip in situ PCR method used reduces the amplification and labeling steps of the sample before the traditional chip detection, and can realize the detection of the four genes in the plasmid pCAMBIA1301 in one step, which greatly shortens the detection time and overcomes the multi-step PCR. It is cumbersome and proposes a feasible method for the comprehensive detection of transgenic rice and transgenic plants with biochips in the future. The specific detection steps are:

[0018] 1. Primer design

[0019] For the conserved sequences of the 35S promoter, reporter genes hpt, aadA, and GUS genes that often appear in transgenic rice, follow the principle that the annealing temperature (Tm value) of each primer is roughly the ...

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Abstract

The present invention is method of performing in-situ PCR reaction on the surface of gene chip to detect the gene to be detected. The upstream primer in common PCR reaction is connected to the arm structure of poly(T), modified 5'-terminal amino radical is connected to aldehyde modified glass slide or other carrier, and the in-situ PCR reaction is performed on the chip. The said method can solve the problems of probe breaking and effective hybridization, and can realize the synchronous processing and in-situ detection. The present invention may be used in SNP detection, transgenic plant detection, disease detection, etc.

Description

technical field [0001] The invention relates to a method for detecting target genes by in-situ polymerase chain reaction on a chip, in particular to a method for detecting target genes by performing in-situ polymerase chain reaction (PCR) on the surface of a gene chip. Background technique [0002] Gene chip refers to the immobilization of a large number of gene probe molecules on a solid support (such as silicon wafer, glass, plastic and nylon membrane, etc.), and then hybridizes with the labeled sample, and the reaction results are analyzed by fluorescence method, enzyme label method and isotope method. detection, and then use scanners and other instruments for data collection, and finally use computer software for data analysis. Using this technology, a large number of gene probes can be fixed on the support at the same time, so a large number of nucleic acid molecules can be detected and analyzed at one time, thereby It solves the shortcomings of traditional nucleic acid...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 赵建龙高秀丽杨剑波景奉香
Owner SHANGHAI INST OF MICROSYSTEM & INFORMATION TECH CHINESE ACAD OF SCI