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Method for distinguishing different plant arising from plant mitochondrion sequence

A mitochondrial and mitochondrial genome technology, applied in the field of biology, can solve the problems of mitochondrial genomes, such as inconvenience, complex and diverse structures, and large differences in changes, and achieve the effects of easy promotion and application, reduced costs, and expanded scope and quantity

Inactive Publication Date: 2004-11-17
浙江省警察学会
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

3) The detection kits used are all imported from abroad, and the price is very expensive
[0005] Because the plant mitochondrial genome sequence has a huge variation, complex and diverse structure (genome is circular, linear; point mutation, deletion, substitution, inversion, etc.), DNA drift, and the existence of plasmids and plasmid-like, which have a great impact on the mitochondrial genome. The sequencing of the genome has brought great inconvenience, so far, there are only a handful of plant mitochondrial genomes published on the Internet (http: / / www.ncbi.nlm.nih.gov)

Method used

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  • Method for distinguishing different plant arising from plant mitochondrion sequence
  • Method for distinguishing different plant arising from plant mitochondrion sequence
  • Method for distinguishing different plant arising from plant mitochondrion sequence

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Experimental program
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Effect test

Embodiment 1

[0024] The extraction of embodiment 1 plant genome DNA:

[0025] Add 20ml of extraction buffer I to a 50ml centrifuge tube and preheat in a 60°C water bath. Rice seedlings or leaves 5-10g, cut into pieces, add liquid nitrogen in a mortar, grind into powder, immediately pour into a preheated centrifuge tube, shake vigorously to mix, keep warm in a 60°C water bath for 30-60min (long time, DNA high yield), shaking occasionally. Add 20ml of chloroform / amyl alcohol / ethanol solution, mix by inversion (gloves are required to prevent damage to the skin), let stand at room temperature for 5-10 minutes, and separate the aqueous phase and the organic phase (re-mix if necessary). Centrifuge at 5000rpm for 5min at room temperature. Carefully pipette the supernatant to another 50ml centrifuge tube, add 1 times the volume of isopropanol, mix well, and leave it at room temperature for a while to form flocculent DNA precipitates. Add 1ml TE to 1.5ml eppendorf. Use a hooked glass rod to rem...

Embodiment 2

[0026] The extraction of embodiment 2 plant mitochondrial genomes:

[0027] Required reagents

[0028] STE buffer: 400 mM sucrose, 50 mM Tris (pH7.8), 20 mM EDTA, 0.2% bovine serum albumin, 0.2% β-mercaptoethanol.

[0029] ST buffer: 400 mM sucrose, 50 mM Tris (pH 7.8), 20 mM EDTA, 0.1% bovine serum albumin.

[0030] TEN buffer: 100 mM Tris (pH 7.2), 50 mM EDTA, 100 mM NaCl and 0.2% β-mercaptoethanol.

[0031] NETF buffer: 1.25M NaCl, 50 mM EDTA, 50 mM Tris (pH 8.0), 50 mM NaF.

[0032] 1M magnesium acetate

[0033] 5M ammonium acetate

[0034] 5M potassium acetate

[0035] 20% SDS

[0036] 1M EDTA

[0037] Phenol (saturated with Tris (pH8.0))

[0038] Phenol:chloroform=1:1

[0039] Chloroform: isoamyl alcohol (24:1)

[0040] 96% and 70% ethanol

[0041] Isopropanol

[0042] DNase

[0043] (1) Separation of mitochondria

[0044] 1) Prepare STE buffer. Add BSA and β-mercaptoethanol.

[0045] 2) Rinse the sample (0°C), and the subsequent steps are all required to...

Embodiment 3

[0070] Embodiment 3 uses primers to carry out PCR amplification

[0071] Reagents required:

[0072] ①10×PCR reaction buffer: 500mmol / L KCl, 100mmol / L Tris·Cl, at 25°C, pH 9.0, 1.0% Triton X-100.

[0073] ② MgCl 2 : 25mmol / L.

[0074] ③ 4 kinds of dNTP mixture: each 2.5mmol / L. That

[0075] ④Taq DNA polymerase 5U / μl.

[0076] ⑤ T4 DNA ligase and ligation buffer.

[0077] ⑥ pUC plasmid digested with Sma I and added with dT.

[0078] ⑦Other reagents: mineral oil (paraffin oil), 1% agarose, 5×TBE, phenol:chloroform:isoamyl alcohol (25:24:1), absolute ethanol and 70% ethanol.

[0079] PCR reaction steps

[0080] ①Mix the following reagents in sequence

[0081] 35 μl H 2 o

[0082] 5 μl 0× PCR reaction buffer

[0083] 4μl 25mmol / L MgCl 2

[0084] 4 μl of 4 dNTPs

[0085] 0.5 μl upstream primers (Table 1)

[0086] 0.5 μl downstream primers (Table 1)

[0087] 0.5μl template DNA (about 1ng)

[0088] Centrifuge for 5 seconds after mixing.

[0089] ②Heat the mixture at ...

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Abstract

The invention discloses a method for distinguishing different plant arising from plant mitochondrion sequence which comprises the steps of, extracting plant genome DNA and mitochondria genome DNA, searching biological mitochondria genome sequence or gene order of several modes from the internet websites, carrying sequence comparison by sequence analyzing software, digging conservative sequences, designing a series of primers by utilizing a primer design software, using the extracted genome DNA and mitochondria DNA as template to carry out polymerase chain reaction to the designed primers, using agar gel electrophoresis for result detecting and analysis to the polymerase chain reaction.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for distinguishing different plants derived from plant mitochondrial sequences. Background technique [0002] Since the 1960s when Nass M. and others directly observed the filamentous DNA in mitochondria with an electron microscope (Nass M M K., et al. 1963), until the late 1980s and early 1990s, people have studied mitochondrial DNA ( The research on the structure and genetic characteristics of mitochondrial DNA (mtDNA) is more in-depth. The mtDNA analysis method has penetrated into the traditional research fields of classification, phylogenetic evolution, population genetics, forensic science and anthropology, and has gradually become an important research tool in these fields. Due to the limitations of technical conditions, the early application of mtDNA was mainly to analyze mtDNA restriction fragment length polymorphism (RFLP). Si...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N27/447
Inventor 徐林苗丁宏徐春法应宗敏朱虹辉吴微微张幼芳姜玉新董海涛
Owner 浙江省警察学会
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