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Single primer capable of multi-species molecular marking and marking method thereof

A technology of molecular markers and marker methods, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., and can solve the problems of undisclosed multi-species molecular marker analysis, poor repeatability of primer amplification, and primer annealing temperature. Low-level problems, to achieve the effect of convenient molecular marker-assisted selection breeding, rich amplification bands, and rich polymorphisms

Active Publication Date: 2017-06-30
广西壮族自治区农业科学院经济作物研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology provides technical benefits such as simplifying the process by providing specific primers with unique sequences within certain areas (called introns) which help reduce costs while still being effective at generating multiple types of DNA fragments called amplicles during different stages of cellular growth cycles. Additionally, this technique allows for precise identification of each type of nucleic acid sequence through its own characteristic properties - including size, structure, chemical composition, etc., making it suitable for studying complex biological systems without relying solely upon any particular features associated therewith.

Problems solved by technology

This patented describes techniques related to nucleic acid sequencing and other fields like biochemistry, medicine science, pharmacology, gaming machines, computer networks, data base systems, drug discovery, disease diagnosis, and identification from DNA samples. These advancements include improved accuracy, efficiency, sensitivity, adaptable assay formats, multiplex signatures, mutation recognition, qPC tagging, and others. Overall, it provides new tools for studying complex mixtures of DNA with specific functions within each species being studied.

Method used

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  • Single primer capable of multi-species molecular marking and marking method thereof
  • Single primer capable of multi-species molecular marking and marking method thereof
  • Single primer capable of multi-species molecular marking and marking method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: In this example, peanuts are used as samples to verify the effectiveness of single primer amplification and screening experiments

[0085] 1. Sample material: Guihua 911 (temporary name), a new peanut strain.

[0086]2. Peanut genomic DNA extraction: Take 0.3-0.5g of fresh peanut leaf samples, cut them into pieces with scissors, put them into a mortar and add 1g of PVP to the mortar, pour enough liquid nitrogen into the mortar several times, and put The leaf samples were fully ground into powder, and all the powder samples were transferred to a 2ml centrifuge tube that had been preheated at 65°C with 1.0ml of CTAB extract in advance, then added 20μl of β-mercaptoethanol, and incubated in a water bath at 65°C for 60 minutes. Mix upside down so that the DNA can be fully separated; then centrifuge at 12000rpm in a refrigerated centrifuge for 10 minutes to remove the bottom residue, draw the supernatant to another 2ml centrifuge tube, and add 0.5μl of RNaseA to t...

Embodiment 2

[0092] Example 2: In this example, sugarcane is used as a sample to verify the effectiveness of single primer amplification and screening experiments

[0093] 1. Sample material: sugarcane variety New Taiwan Sugar 22.

[0094] 2. Sugarcane genomic DNA extraction: Take 0.3-0.5g of fresh sugarcane core leaves, cut them into pieces with scissors, put them into a mortar and add 1g of PVP to the mortar, pour enough liquid nitrogen into the mortar several times, and put the leaves The sample is fully ground into powder, and all powder samples are transferred to a 2ml centrifuge tube that has been preheated at 65°C with 1.0ml of CTAB extract in advance, then added 20μl of β-mercaptoethanol, and warmed in a water bath at 65°C for 60 minutes. Invert and mix well so that the DNA can be fully separated; then centrifuge in a refrigerated centrifuge at a speed of 12000rpm for 10 minutes to remove the bottom residue, draw the supernatant to another 2ml centrifuge tube, and add 0.5 μl of RNa...

Embodiment 3

[0097] Example 3: In this example, rice is used as a sample for molecular marker analysis

[0098] 1. Sample material: 16 rice varieties, namely: Kasalah, Baipi Nuogu, Wandaonuo, Huagu, Taiwanbai, Xigu, Huangbaizhan, Guanghui 998, Nipponbare, Huanghongnuo, Xiangnuo, Shanjaponica , Malay, Huapi, Late Upland Rice and Rongbaohong.

[0099] 2. Genomic DNA extraction: Take 0.3-0.5g of fresh rice tender leaves, cut them into pieces with scissors, put them into a mortar and add 1g of PVP to the mortar, pour enough liquid nitrogen into the mortar several times, and the leaf samples Fully grind into powder, transfer all powder samples to a 2ml centrifuge tube that has been preheated at 65°C with 1.0ml of CTAB extract in advance, then add 20μl of β-mercaptoethanol, and warm it in a water bath at 65°C for 60 minutes, during which time it is turned upside down Mix well so that the DNA can be fully separated; then centrifuge at 12000rpm in a refrigerated centrifuge for 10 minutes to remov...

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Abstract

The invention relates to the field of biotechnology, especially to a single primer capable of multi-species molecular marking and a marking method thereof. The primer is designed according to the AT-rich intron region and regulatory region of a gene in a eukaryotic genome, and primer sequence length is 17 bp. A filling sequence is any sequence rich in GC bases; a core-region sequence is formed by random arrangement of AT bases; a selective base sequence is any base sequence. The primer can be used among eukaryote. Thus, synthesis cost of the primer is reduced, and utilization rate of the primer is greatly raised. Meanwhile, the DNA molecular marking method is based on a PCR reaction, polymorphism begins with intron sequence mutation and insertion/deletion. The method is simple and efficient; polymorphism is high; there are abundant amplified bands; results are reliable; and the bands are easy for separation and sequencing.

Description

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Claims

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Application Information

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Owner 广西壮族自治区农业科学院经济作物研究所
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