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Method for preparing internal standard of molecular weight, and internal standard of molecular weight prepared by using the method

A molecular weight internal standard and fluorescent marker technology, which is used in biochemical equipment and methods, DNA preparation, and microbial determination/inspection, etc. Efficiency, easy operation and time saving effect

Active Publication Date: 2007-10-10
DINGSHENG TECH BEIJING +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is difficult to obtain a large number of accurate fluorescence-labeled molecular weight internal standards by conventional methods for preparing molecular weight standards

Method used

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  • Method for preparing internal standard of molecular weight, and internal standard of molecular weight prepared by using the method
  • Method for preparing internal standard of molecular weight, and internal standard of molecular weight prepared by using the method
  • Method for preparing internal standard of molecular weight, and internal standard of molecular weight prepared by using the method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Preparation of molecular weight internal standard

[0058] 1.1 Design upstream primers and synthesize ROX-labeled upstream primers.

[0059] The DNA template used was pUC18 plasmid (Takara Company, Cat. No. D3218). The 5'GCGAAACCCGACAGGACTA 3' at 927bp was selected as the upstream primer, and the annealing temperature was 58°C. ABI 394 nucleic acid synthesizer synthesized primers, and labeled with ROX at the 5' of the primers, and purified the primers by HPLC.

[0060] 1.2 Design downstream primers according to the fragment length.

[0061] Select a series of 16 fragments ranging in length from 70, 80, 100, 120, 140, 160, 180, 200, 240, 280, 320, 360, 400, 450, 490, 500. It can also be a plurality of nucleotide fragments with different lengths between 927-1425bp. Primers were synthesized by ABI 394 nucleic acid synthesizer and purified by PAGE. The downstream primer selection sequence is as follows:

[0062] Primer name Primer sequence (5′-3′)

[0063] ...

Embodiment 2

[0094] Example 2. Analyze gene fragments and check accuracy with ROX-labeled molecular weight internal standard

[0095] The allele ladder (one of the Goldeneye 16A identification kit components of Basic Cognitive Technology (Beijing) Co., Ltd.) with a length of 100-480bp marked by FAM, HEX, and TMR was used for molecular weight calibration. Containing 210 allelic fragments, calculate the average size and variance of each fragment. In the control experiment, the product ILS 600 (product number DG2611) of Promega Company of the United States was used to analyze the allelic ladder. ILS 600 is composed of 22 double-stranded DNAs of 60-600bp labeled by ROX, in which the 60-200bp fragments are separated by 20bp, the 200-500bp fragments are separated by 25bp, and the 500-600bp fragments are separated by 50bp.

[0096] The electrophoresis detection was completed on the ABI 3100 genetic analyzer, the electrophoresis voltage was 15kV, and the collection time was 1400 seconds. The sam...

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Abstract

This invention relates to a method for preparing molecular weight internal standard. The method comprises: immobilizing upstream primer 5'GCGAAACCCGACAGGACTA3' with pUC18 plasmid as the template, designing downstream primers with a certain distance, and proliferating to obtain multiple nucleotide segments. The method obtains a series of segments with different lengths by immobilizing the upstream primer and designing the downstream primers in sequence. The traditional method for synthesizing primers by fluorescent labeling has such disadvantages as high cost and short preservation time. The method in this invention synthesizes the upstream immobilized primer only, which can largely lower the primer synthesis cost and production cost. Since the upstream primer is the same, the lengths and annealing temperatures of the downstream primers are similar, and the proliferation conditions for all segments are identical, which can facilitate easy operation (all proliferations are performed on the same gene proliferation apparatus with the same reaction program), saved time, high working efficiency, and large-scale production.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a molecular weight internal standard and a preparation method thereof. Background of the invention [0002] Gene scanning and genotyping technologies have developed rapidly since the beginning of the Human Genome Project. It is widely used in pathogenic gene screening, disease gene linkage and association typing, early diagnosis of tumors and other diseases, and forensic individual identification. An important means of genotyping technology is to use DNA sequencer or genetic analyzer to analyze the molecular weight of gene fragments. When analyzing the molecular weight of this type of instrument, a molecular weight standard is needed to calculate the sample, that is, the molecular weight internal standard, also known as the internal molecular weight control (Internal Lane Standard). [0003] Compared with the traditional molecular weight standard (marker), the molecular weight inte...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34C12N15/11C12N15/10
Inventor 高阳陈初光马斌王曙光
Owner DINGSHENG TECH BEIJING
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