Nucleotides sequence, molecule probe and method for identifying zhejiang fritillary variant-dong fritillary

A technology of nucleotide sequence and nucleic acid molecules, which is applied in the field of identification of Fritillaria varietata---East Fritillary. It can solve the problems of research reports and unseen technology for fast identification of Fritillaria fritillaria DNA, and achieve less sample consumption. Effect

Inactive Publication Date: 2008-03-12
ZHEJIANG UNIV
View PDF0 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the identification of Fritillaria species is mainly based on the morphology and chemical composition of medicinal materials, and there is no research report on the rapid identification technology of Fritillaria ternata DNA

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Nucleotides sequence, molecule probe and method for identifying zhejiang fritillary variant-dong fritillary
  • Nucleotides sequence, molecule probe and method for identifying zhejiang fritillary variant-dong fritillary
  • Nucleotides sequence, molecule probe and method for identifying zhejiang fritillary variant-dong fritillary

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of Orient Fritillaria Specific Nucleotide Sequence

[0041] 1 Genomic DNA extraction

[0042] The modified CTAB method (Doyle, 1991) was used to extract genomic DNA from fritillaria leaves, and the steps were as follows:

[0043] (1) Take 0.05 g of silica gel-dried Fritillaria leaf material, put it in a BIO101 tube and grind it into powder, then add 2ml of 2×CTAB extraction buffer (100mM Tris-HCL (PH8.0), 20mM EDTA, preheated at 65°C, 1.4M NaCl, 2% CTAB, 2% β-mercaptoethanol), gently shake to disperse the solution evenly, 65°C water bath for 20-30 minutes, gently shake every 5 minutes during the water bath.

[0044] (2) Cool to room temperature, add an equal volume of chloroform:isoamyl alcohol (24:1), mix well, and centrifuge at 10,000 g for 10 minutes.

[0045] (3) Transfer the upper aqueous phase into a new 5ml centrifuge tube, add 1 / 10 volume of 10×CTAB-0.7MNaCl, mix gently, then add an equal volume of chloroform:isoamyl alcohol (24:1) for e...

Embodiment 2

[0064] Embodiment 2: Preparation of friD1 and friD2 specific nucleic acid molecular probes of Fritillaria fritillaria

[0065] On the basis of obtaining the specific nucleotide sequence of Fritillaria fritillaria, use Primer Primer 5.0 (VinaySingh, 1998) software to design, draw friD1 and friD2 (respectively 79-97bp and 661 bp shown in 1 in the sequence list The nucleotide composition and arrangement of -679bp) are good oligonucleotide fragments for identification of Fritillaria japonica. According to the nucleotide composition arrangement of friD1 and friD2 (the nucleotide composition and arrangement shown in the 2 and 3 sequences in the sequence listing), they were synthesized on an automatic DNA synthesizer.

Embodiment 3

[0066] Embodiment 3: the identification (conventional PCR method) of Eastern Fritillaria

[0067] 1. Extraction of DNA: using the improved CTAB method to extract DNA from Fritillaria

[0068] 2. The PCR reaction system is:

[0069] 10×Buffer(Mg 2+ free)

MgCl 2 (25mM)

dNTPs (10mM)

friD1 (5 μM)

FriD2 (5μM)

Taq enzyme (5U / μl)

Template DNA (30ng / μl)

ddH2O

2.5μl (1×Buffer)

2μl (2.0mM)

0.5μl (0.2mM)

1μl (0.4μM)

1μl (0.4μM)

0.25μl (1.2U)

2.5μl (60ng)

15.76μl

total capacity

25μl

[0070] 3. PCR operation: Take two 0.5 ml PCR tubes, add 22.5 microliters of PCR mixture according to step 2, then add 2.5 microliters of DNA to one tube, and 2.5 microliters of PCR mixture (control) into the other tube, and put On the PCR instrument, carry out the PCR reaction according to the following procedure:

[0071] 94℃ 5min

[0072]

[0073] The results of PCR amplification we...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention discloses a nucleotide sequence, a nucleic acid molecular probe and a method for verifying a variant Fritillaria thunbergii.Miq.var. chekiangensis Hsiao et K.C.Hsia, and pertains to the technical field for verifying traditional Chinese medicine. The invention provides a special sequence for expanded ISSR between simple repeated sequences of Fritillaria thunbergii.Miq.var. chekiangensis Hsiao et K.C.Hsia and the verifying probe and method based on the sequence for the Fritillaria thunbergii.Miq.var. chekiangensis Hsiao et K.C.Hsia. The benefits with the invention are: 1) less sample use. The whole operation can be completed with few sample; 2) high accuracy and sensibility. The friD1 / friD2 is a special molecular probe for Fritillaria thunbergii.Miq.var. chekiangensis Hsiao et K.C.Hsia, and the probe will have negative reaction for other species.

Description

technical field [0001] The invention belongs to the technical field of using molecular biological methods to identify a variety of Fritillaria fritillaria---East Fritillaria, in particular, it relates to a nucleotide sequence, a nucleic acid molecular probe and a method for identifying a variety of Fritillaria japonica. Background technique [0002] Fritillaria thunbergii Miq.var.chekiangensis Hsiao et K.C.Hsia is mainly produced in Dongyang, Pan'an and other places in Jinhua, Zhejiang Province, and has a long history of cultivation. Bitter and cold in nature and taste, it has the effects of clearing away heat, resolving phlegm, dispelling stagnation and dispelling stagnation. It is used for wind-heat, phlegm-fire cough, lung abscess, breast abscess, scrofula, sore, heart and chest depression, etc. The efficacy and indications are similar to those of Fritillaria fritillata . The 94th edition of "Zhejiang Province Traditional Chinese Medicine Processing Standards" recorded i...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/29C12Q1/68
Inventor 赵云鹏刘晓贤陈川潘兰兰陈斌龙傅承新
Owner ZHEJIANG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap