Multiplex fluorescence PCR (polymerase chain reaction) universal adapter for microsatellite detection, and detection method and application thereof

A technology of multiple fluorescent and universal joints, applied in the field of multiple fluorescent PCR, can solve the problems of unusable fluorescent primers, waste of funds and time, etc., and achieve the effects of reducing non-specific amplification, saving funds, and strong versatility

Inactive Publication Date: 2014-12-03
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the synthesized primers cannot be added to the multiplex PCR system during the experiment, the synthesized fluorescent primers cannot be used in subsequent experiments
Determine a set of multiplex PCR primers. Generally, only a part of the fluorescently labeled primers synthesized can be used in experiments, resulting in a waste of money and time

Method used

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  • Multiplex fluorescence PCR (polymerase chain reaction) universal adapter for microsatellite detection, and detection method and application thereof
  • Multiplex fluorescence PCR (polymerase chain reaction) universal adapter for microsatellite detection, and detection method and application thereof
  • Multiplex fluorescence PCR (polymerase chain reaction) universal adapter for microsatellite detection, and detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The multiplex fluorescent PCR universal connectors used in this example, which can be used for microsatellite detection, include 4 connectors, which are respectively connector 1, connector 2, connector 3 and connector 4. The nucleotide sequences of connectors 1-4 are shown as SEQ ID: Shown in 1~4.

[0046] Microsatellite detection is mostly used in eukaryotes, and the selection of universal adapters mainly considers the lowest similarity with the DNA sequence of eukaryotes, so the plasmid sequences of prokaryotic bacteria are selected to design adapters. Bacterial plasmids already have universal primers for cloning and sequencing, and the present invention uses one of the M13 universal primers, ie adapter 1. Use the primer5 software to detect that the annealing temperature of adapter 1 is 51.8°C. In order to make the amplification efficiency of the adapter close, use this as a standard to search for sequences in the entire plasmid sequence to design primers with similar...

Embodiment 2

[0065] The 4 multiplex fluorescent PCR universal connectors used in this example are the same as those in Example 1.

[0066] The method provided in this example for multiplex PCR amplification using the above-mentioned multiplex fluorescent PCR universal joint that can be used for microsatellite detection and its application in allelic amplification and separation in the multiplex PCR system of the yellow-throated aquatic turtle are as follows:

[0067] (1) Sampling

[0068] Extract 10-20 μL of yellow-throated aquatic turtle blood, put it in a centrifuge tube filled with 200 μL buffer TL (MicroElute Genomic DNA Kit, OMEGA, USA), and store in an ice box (for DNA extraction on the same day);

[0069] (2) Extraction of genomic DNA

[0070] Genomic DNA was extracted with the MicroElute Genomic DNA Kit kit, 1% agarose gel electrophoresis was used to detect the integrity of the DNA, and the DNA was subjected to NanoQ TM The micro-spectrophotometer (Boao) detects the concentratio...

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Abstract

The invention discloses a multiplex fluorescence PCR (polymerase chain reaction) universal adapter for microsatellite detection, which comprises an adapter 1, an adapter 2, an adapter 3 and an adapter 4, wherein the nucleotide sequences of the adapters 1-4 are disclosed as SEQ ID:1-4. The universal adapter has the advantages of high PCR amplification efficiency and no interference, and can not interfere with microsatellite primers to be amplified. The invention also discloses a method for carrying out microsatellite multiplex fluorescence PCR detection by using the multiplex fluorescence PCR universal adapter for microsatellite detection. In the multiplex PCR process, primer screening and typing experiments can be performed only after the four or three fluorescence adapters are synthesized. The method has the advantages of simple steps, short experimental period and low experimental cost.

Description

technical field [0001] The invention belongs to the technical field of multiple fluorescent PCR, and in particular relates to a multiple fluorescent PCR universal joint that can be used for microsatellite detection, and a method and application for using the same to perform microsatellite multiple PCR detection. Background technique [0002] Microsatellite (Microsatellite), also known as Simple Sequence Repeat (SSR), Short tandem repeats (Short tandem repeats), Simple Sequence Length Polymorphism (Simple sequence length polymorphism), it is a fragment in the DNA molecule, with The 1-6bp nucleotide sequence (called the core sequence, coresequence) is a highly repetitive sequence that is evenly distributed in the entire genome in the form of end-to-end tandem repeats. Generally tens to hundreds of bp long. They are widely distributed in various eukaryotic genomes, and the distribution is relatively uniform. The alleles of microsatellites have fast mutation, high polymorphism...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6855C12Q2525/191
Inventor 赵建朱新平李伟王亚坤文萍
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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