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High throughput single nucleotide polymorphism assay

A technology of oligonucleotides and fluorescent dyes, applied in the field of high-throughput single nucleotide polymorphism assays, can solve the problems of expensive, difficult and time-consuming high-throughput detection

Inactive Publication Date: 2014-08-13
AGRI GENETICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, high-throughput detection of the HaAHASL1-A122(At)T-specific mutation is made difficult by the presence of paralogous sequences that share a high level of sequence similarity with HaAHASL1
Existing assays for the detection of the HaAHASL1-A122(At)T mutation, especially gel electrophoresis-based assays, are low-throughput, inconvenient, time-consuming, and expensive

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0068] Example 1: DNA extraction and quantification

[0069]A method for detecting the HaAHASL1-A122(At)T mutant allele was developed using plants from a sunflower line previously characterized as homozygous for the HaAHASL1-A122(At)T mutant allele. In addition, a method to detect the HaAHASL1-A122(At)T wild-type allele was developed using another sunflower line previously characterized as homozygous for the HaAHASL1-A122(At)T wild-type allele. As a result, a HaAHASL1 method consisting of a homogeneous assay detection system for a PCR process utilizing FRET was developed. After the development of the HaAHASL1 method consisting of a homogeneous assay detection system for the PCR process using FRET, it was validated using previously genotyped sunflower lines.

[0070] Genomic DNA was extracted from leaf tissues of sunflower lines homozygous for the HaAHASL1-A122(At)T mutant allele and a sunflower line homozygous for the HaAHASL1 wild-type allele using the QiaGene DNeasy96 Plant...

Embodiment 2

[0071] Example 2: Primer Design

[0072] Three primers were manually designed based on the nucleotide sequence information of HaAHASL1 (SEQ ID NO:1) and HaAHASL1-A122(At)T mutant allele (SEQ ID NO:2) (Table 1).

[0073] A mutant allele detection common primer 043-0001.1.A1 (SEQ ID NO: 3) containing a tail at the 5' end was synthesized, and the sequence of the tail was the same as the fluorescently labeled primer of the KBiosciences reaction kit. KBiosciences Reaction Kit Fluorescently Labeled Primers are labeled with the fluorescent dye 5-carboxyfluorescein (FAM). This primer is specifically designed to amplify the HaAHASL1-A122(At)T mutant allele.

[0074] A wild-type allele detection common primer 043-0001.2.A2 (SEQ ID NO: 4) containing a tail at the 5' end that is the same as the fluorescently labeled primer of the KBiosciences reaction kit was synthesized. KBiosciences Reaction Kit Fluorescently Labeled Primers are labeled with the fluorescent dye 2’,7’-dimethoxy-4’,5’-d...

Embodiment 3

[0077] Example 3: HaAHASL1 single nucleotide polymorphism assay

[0078] The three HaAHASL1 common primers were mixed to achieve the concentrations described in Table 2. The HaAHASL1 reaction cocktail and thermal cycling program are described in Tables 3 and 4, respectively. exist HaAHASL1 PCR reactions were performed in a 96-well plate format on a PCR System 9700 (Applied Biosystems, Carlsbad, CA). HaAHASL1 PCR reaction products were read on a fluorescent microplate reader using the following parameters: (1) FAM: excitation at about 485 nm; emission at about 535 nm; and (2) JOE: excitation at about 525 nm, emission at about 560 nm. Fluorescence readout data were saved in Microsoft Excel format and the data were plotted on the x-y axis. The FAM values ​​are plotted on the X-axis and the JOE values ​​are plotted on the Y-axis.

[0079] figure 1 The results of the assay are presented in . Samples known to be homozygous for the HaAHASL1 wild-type allele are clustered in th...

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Abstract

A method consisting of a homogeneous assay detection system for a PCR process using FRET for detection and zygosity analysis of the HaAHASL1-A122(At)T single nucleotide polymorphism in sunflower is provided. The method provides specific sunflower-genome primers that can be used to detect the presence or absence of the HaAHASL1-A122(At)T single nucleotide polymorphism. The primer combinations for use in an endpoint PCR assay capable of determining zygosity and for assisting in breeding introgression are described.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application 61 / 564,464, filed November 29, 2011, which is expressly incorporated herein by reference. [0003] Citing an Electronically Submitted Sequence Listing [0004] An official copy of the Sequence Listing was submitted electronically via EFS-Web as an ASCII formatted Sequence Listing in a file named "69826USNP_SEQ_ID_ST25" created on November 27, 2012 and having a size of 2kb and simultaneously with the description. The Sequence Listing contained in this ASCII formatted file is part of the specification and is hereby incorporated by reference in its entirety. Background of the invention [0005] The present disclosure focuses on a method consisting of a homogeneous assay detection system using a PCR process using FRET for the detection of the AHASL1-A122(AT)T single nucleotide polymorphism in sunflower (Helianthus annuus L). The AHASL1-A122(AT)T allele confe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53C12Q1/68C12N15/82
CPCC12Q2600/13C12Q1/6895C12Q2600/156C12Q1/6827C12Q1/6858C12Q2535/125C12Q2563/107C12Q2531/113
Inventor W.高S.P.库姆帕特拉R.M.本森J.T.格迪斯
Owner AGRI GENETICS
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