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Wide-space high-throughput amplification method applicable to gene detection

A gene detection, high-throughput technology used in WideSpace amplification

Inactive Publication Date: 2012-04-04
INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is impossible to detect multiple genes simultaneously, so a high-throughput amplification method is necessary

Method used

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  • Wide-space high-throughput amplification method applicable to gene detection
  • Wide-space high-throughput amplification method applicable to gene detection
  • Wide-space high-throughput amplification method applicable to gene detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, a kind of WideSpace high-throughput amplification method suitable for gene detection comprises the following steps:

[0021] (1) Set the target gene and the number n of target genes. According to the number n of target genes and the resolution of the electrophoresis system, reasonably allocate the length span of the fragment size of the final product: for the electrophoresis system with a resolution between 10 bp and 20 bp, the length span of the product fragments of each target gene is 15 bp ~25 bp; for the resolution of the electrophoresis system between 50 bp and 100 bp, the product fragment length of each target gene spans between 60 bp and 110 bp.

[0022] (2) Design of target gene primers, universal amplification primers, and nested primers: a) Design target gene primers according to the general principles of primer design; in order to ensure the success of subsequent detection, the GC% content of all upstream and downstream primers is required to be...

Embodiment 2

[0027] Example 2, if the detection object is RNA, a separate reverse transcription reaction needs to be performed in advance before step (3) of Example 1. 20~25 uL reverse transcription reaction solution contains: 1x reverse transcription buffer (50 mmol / L Tris-Hcl, 75mmol / L KCl, 1.5~5.0mmol / L MgCl 2 , 10mmol / L DTT, pH8.3(25℃)), 0.5mmol / L dNTP (including A, G, C, T), 20U RNase inhibitor, 1~2 U reverse transcriptase, 0.1~0.5 umol / L Downstream nested primers, 100-500 ng sample RNA, react according to parameters 40-55°C, 15-60 min, 85°C, 5 min. After the reaction is completed, the detection reaction in step (3) of Example 1 is carried out. The molar concentration of each substance is the final concentration of the solution.

Embodiment 3

[0028] Example 3, (1) Assumed detection objects are: Gene1, Gene2, Gene3, Gene4, Gene5, a total of five genes.

[0029] (2) According to the general rules of primer design, the GC% content of the primers is between 55% and 60%, and the TM value is not less than 58°C. The target gene-specific primers are obtained as follows:

[0030] Gene1

[0031] Upstream: 5'-TCACCTGTATCTTGAATTATG-3'

[0032] Downstream: 5'-GTACAACTATAGCAGAATCTT-3';

[0033] Gene2

[0034] Upstream: 5'-TAGACGCAGGTTGAAGAT-3'

[0035] Downstream: 5'-GAGTGAGGATAGCATACGA-3';

[0036] Gene3

[0037] Upstream: 5'-CCACCTGCAGCCGATAACACCTC-3'

[0038] Downstream: 5'-GGTGGAATTGGCTCTTCCTGCGTTT-3';

[0039] Gene4

[0040] Upstream: 5'-CAGTGGAGTGGAAGGAGAA-3'

[0041] Downstream: 5'-ATTACCGTGCCTGTTGGA-3';

[0042] Gene5

[0043] Upstream: 5'-CGTACGTGCCGGCGACCATT-3'

[0044] Downstream: 5'-GCTTGGGCGACCACGGGAAT-3'.

[0045] The design of the internal reference gene IPC was the same as that of the above-mentioned g...

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Abstract

The invention discloses a wide-space high-throughput amplification method applicable to gene detection. The method comprises the following steps of: designing a target gene primer, a universal amplification primer and a nested primer; after the universal amplification primer is designed, respectively adding the upstream of the universal amplification primer to the upstream primer 5' end of each target gent and respectively adding the downstream of the universal amplification primer to the downstream primer 5' end of each target gent to obtain the nested primer. The invention overcomes the defect that the conventional gene detection method is difficult to complete the synchronous detection of more than five gene targets within one reaction. The wide-space high-throughput amplification method provided by the invention can be introduced to transgene detection, pathogenic microorganism detection and screening, polygene expression analysis and other related fields.

Description

technical field [0001] The invention belongs to the field of gene detection by molecular biological methods, in particular to a WideSpace amplification method for high-throughput parallel detection of 10 or more genes. Background technique [0002] Conventional gene detection technology generally uses PCR to amplify and detect genes, the throughput is low, and the number of target genes generally does not exceed 5. It is impossible to detect multiple genes at the same time, so a high-throughput amplification method is necessary. The method of the present invention is mainly based on adding a pair of universal amplification primers to the two ends of the target gene-specific primers, and on the basis of the specific amplification of the target gene primers, the amplification primers are used to uniformly amplify the signal, thereby realizing multiple target genes. high-throughput parallel detection. Contents of the invention [0003] The purpose of the present invention i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王昱王国民肖进文聂福平杨俊李应国
Owner INSPECTION & QUARANTINE TECH CENT OF CHONGQING ENTRY EXIT INSPECTION & QUARANTINE BUREAU