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Pleuronectiformes soleidae fish ribosome ITS1 (internal transcribed spacer region 1) universal primer design method and application

A technique of internally transcribed spacers and universal primers, applied in biochemical equipment and methods, microbe measurement/inspection, DNA/RNA fragments, etc., can solve blank and rare problems, achieve high accuracy, fast evolution rate, Addressing the Effects of Germplasm Mixing

Inactive Publication Date: 2018-04-06
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Soleidae is the most abundant group of flatfishes (about 35 genera and 175 species), and many species have very important economic value, but so far the reports on the ribosome sequence of Soleidae fish are extremely rare. The study of transcriptional spacer 1 is blank

Method used

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  • Pleuronectiformes soleidae fish ribosome ITS1 (internal transcribed spacer region 1) universal primer design method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] A method for designing general primers for ribosomal transcribed spacer 1 (ITS1) in Pleuriformes Soleidae fishes, including the following steps:

[0018] 1) Obtain the 18S rDNA and 5.8S rDNA sequences of Soleidae from Genbank, use ClustalXv1.83 to compare the sequences, and find the conserved sequences at the 3' end and 5' end, respectively, as primer candidate regions;

[0019] 2) Use the OLIGO v6 software to analyze the sequence of the candidate region. The selection conditions for the primer sequence: the primer length is about 23-25bp, the GC content is 40%-65%, and the annealing temperature is 55-60°C.

Embodiment 2

[0021] Sequence determination of ribosomal transcribed spacer 1 (ITS1) in Pleuriformes Soleidae fish, the method is as follows:

[0022] 1) Genomic DNA extraction: using the standard phenol-chloroform method to extract the moth-eyebrow sole, striped sole, round-scaled sole, oval sole, Japanese hook-billed sole, eye-spotted leopard sole, brown-spotted ctenophore sole, and Sclerophyllus carinii Sole, Genomic DNA of Sole Senegal, dissolved in TE, stored at -20°C;

[0023] 2) Amplification reaction: use the extracted genomic DNA as a template to carry out the amplification reaction of ribosomal transcribed spacer 1 (ITS1), take 100ng of template DNA, 0.5 μL of upstream primers with a concentration of 10 μmol / L, and 0.5 μL of upstream primers with a concentration of 10 μmol / L downstream primers, 2.5 μL 10×buffer, 0.5 μL dNTP with a concentration of 10 mmol / L, 1.5 μL MgCl with a concentration of 25 mmol / L 2 , mixed with 0.2 μL Taq polymerase, added double distilled water to 25 μL, ...

Embodiment 3

[0035] Application of universal primers for ribosomal transcribed spacer 1 (ITS1) in Soleidae fishes of the order Soleidae in the identification of Soleidae fishes, the specific process is as follows:

[0036] 1) Extraction of genomic DNA: extract the genomic DNA of the Soleidae fish to be identified by the standard phenol-chloroform method, dissolve in TE, and store at -20°C;

[0037] 2) Amplification reaction: use universal primers to amplify the extracted DNA in ribosomal transcribed spacer 1 (ITS1), take 100ng of template DNA, 0.5 μL of upstream primers with a concentration of 10 μmol / L, and 0.5 μL of upstream primers with a concentration of 10 μmol / L downstream primers, 2.5 μL 10×buffer, 0.5 μL dNTP with a concentration of 10 mmol / L, 1.5 μL MgCl with a concentration of 25 mmol / L 2 , mixed with 0.2 μL Taq polymerase, added double-distilled water to 25 μL, and the amplification reaction was carried out on a PTC-200 PCR instrument. Extend for 1 min at °C for 30 cycles, and ...

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Abstract

The invention discloses a pleuronectiformes soleidae fish ribosome ITS1 (internal transcribed spacer region 1) universal primer design method and application. The method has the beneficial effects that the disclosed primer design method is designed on the basis of the conservative 18S rDNA and 5.8 S rDNA sequences at the two ends of ribosome ITS1; the obtained primer has very high specificity; theamplified ITS1 evolutionary rate is relatively high; similarity basically shows within the variety; the obvious differences are shown between varieties; when the primer is applied to soleidae fishes,the accuracy is high; the form feature identification defects are overcome; the problem of idioplasm mixing is solved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a design method and application of a universal primer for the internal transcriptional spacer 1 (ITS1) of fish ribosomes of the order Soleidae. Background technique [0002] Soleidae has an oval body with extremely flattened sides. Both eyes are located on the right side of the body, and some are located on the left side. The mouth is small and asymmetrical. The upper and lower jaws are underdeveloped. The teeth of the operculum are more developed, with fine tooth bands, the palatine bone has no teeth, and the edge of the preopercle bone is covered with a membrane or scales. The body of the fish is covered with round scales, ctenoid scales or membranes. The lateral line is single. The dorsal fin starts above the eyes. Some of the dorsal, anal and caudal fins are connected, while others are separated. The pectoral fins are small or absent, and the pelvic fin...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888
Inventor 龚理孔晓瑜
Owner ZHEJIANG OCEAN UNIV
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