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Method and kit for directly amplifying DNA (deoxyribonucleic acid) bar code of trace sample without extraction

A very small, extraction-free technology, applied in the biological field, can solve the problems of inability to achieve accurate species identification, insufficient effective data, etc., and achieve the effects of high reliability and adaptability, high amplification efficiency and good stability.

Active Publication Date: 2013-12-11
岳巧云
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the serious shortage of effective data in the current database, DNA barcode technology often cannot achieve the purpose of accurate species identification in practice. Therefore, the data in the DNA barcode database needs to be supplemented urgently. For the establishment of DAN barcode data, voucher samples are the source samples of DNA data. It is very important for the verification of data and specimens in the future. In order to ensure the morphological integrity of the voucher specimens, when taking tissues, try to reduce the size of the tissue blocks

Method used

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  • Method and kit for directly amplifying DNA (deoxyribonucleic acid) bar code of trace sample without extraction
  • Method and kit for directly amplifying DNA (deoxyribonucleic acid) bar code of trace sample without extraction
  • Method and kit for directly amplifying DNA (deoxyribonucleic acid) bar code of trace sample without extraction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: DNA template amplified DNA barcode sequence prepared by different concentrations of lysate

[0025] Prepare 100 ml of NaOH solutions with concentrations of 10, 25, 50, 75, and 100 mM respectively, and autoclave them for later use.

[0026] Preparation of buffer: 1.22g Tris, 7.46g KCl, 0.3g EDTA plus deionized water to make up to 100ml, adjust the pH to 9.5, autoclave for later use.

[0027] Take 1 / 5 hind tarsus (about 10 μg) of five flies and put them into five 1.5ml centrifuge tubes, add liquid nitrogen, grind them into powder with a grinding rod, add 10 and 25 μg to the five centrifuge tubes respectively , 50, 75, and 100 mM NaOH solution 180 μl, placed at 95°C for 10 minutes, then briefly centrifuged at 4000 rpm for 5 seconds, then added 20 μl of buffer solution, placed at 95°C for 10 minutes, centrifuged at 12,000 rpm for 1 minute, discarded the precipitate, and the obtained supernatant was the fly DNA solution.

[0028] Using the fly DNA solution prepar...

Embodiment 2

[0032] Example 2: Preparation of DNA Template Amplified DNA Barcode Sequence by Mixing Buffer and Lysis Solution in Different Volume Ratio

[0033] The preparation method of lysate and buffer is the same as in Example 1.

[0034] Take 1 / 5 hind tarsus (about 10 μg) of five flies and put them into five 1.5ml centrifuge tubes respectively, add liquid nitrogen, grind them into powder with a grinding rod, and add 50 mM NaOH to the five centrifuge tubes respectively Solution 20, 60, 120, 180, 200μl, placed at 95°C for 10min, centrifuged briefly at 4000rpm for 5s, then added 180, 140, 80, 20, 0μl of buffer respectively, placed at 95°C for 10min, centrifuged at 12000rpm for 1min, discarded the precipitate, and obtained The supernatant is the fly DNA solution.

[0035] The PCR amplification reaction system and reaction conditions were the same as in Example 1.

[0036] Amplification results such as figure 2 As shown, the negative control is ddH 2 O is the result of template amplif...

Embodiment 3

[0038] Example 3: Amplification of DNA barcode sequences at different annealing temperatures

[0039] The preparation method of lysate and buffer is the same as in Example 1.

[0040] Take 16 fly 1 / 5 hind tarsus (about 10μg) and put them into 16 1.5ml centrifuge tubes, add liquid nitrogen, grind them into powder with a grinding rod, and then add 50mM NaOH solution to the centrifuge tubes respectively 180 μl, placed at 95°C for 10 minutes, then briefly centrifuged at 4000rpm for 5s, then added 20μl of buffer solution, placed at 95°C for 10 minutes, centrifuged at 12000rpm for 1min, discarded the precipitate, and the obtained supernatant was the fly DNA solution.

[0041] The PCR amplification reaction system was the same as in Example 1.

[0042] The annealing temperatures of the 16 samples were 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, and 62°C, respectively.

[0043] Other amplification reaction conditions are the same as in Example 1.

[0044] Amplificat...

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Abstract

The invention discloses a method and kit for directly amplifying a DNA (deoxyribonucleic acid) bar code of a trace sample without extraction. The method comprises the following steps: adding a lysis solution and a buffer solution in a volume ratio of 1:(0-9) to obtain a trace sample DNA solution, and carrying out (polymerase chain reaction) amplification by using a forward primer LCO1490 and reverse primer HCO2198 to obtain the DNA bar code sequence of the trace sample. By optimizing the formula and the ratio of the lysis solution and the buffer solution and optimizing the reaction system and reaction conditions, the invention can avoid mixed extraction of template DNAs on a plurality of mini animal individuals, thereby ensuring the singleness of the DNA template source. The method disclosed by the invention has the advantages of high favorable, high amplification efficiency, high reliability and wide adaptability.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a method and a kit for directly amplifying DNA barcodes of very small samples without extraction. Background technique: [0002] Mites are important medical vectors, which spread many diseases and pose a great threat to human health. The individuals of mites are very small, usually less than 1mm. It is difficult to obtain template DNA with a high enough concentration using existing DNA extraction methods or commercial kits, and it is difficult to perform subsequent PCR amplification of specific genes. Individuals or even dozens of individuals are mixed together to extract genomic DNA, which cannot guarantee the singleness of the source of template DNA, which will cause great trouble for subsequent molecular analysis. Therefore, it is necessary to develop a simple and efficient one. A method and a kit for directly amplifying a DNA barcode sequence from a single individua...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 岳巧云胡佳邱德义陈健刘德星魏晓雅吴可量刘国雄
Owner 岳巧云
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