CN1144880C - Biological variety genome DNA fingerprint atlas - Google Patents

Abstract

The present invention relates to a biological variety genome DNA fingerprint diagram spectrum. The diagram spectrum uses the molecular genetic marking technique for constructing genome DNA fingerprint diagram spectrums of biological species comprising persons, plants, animals, microorganisms, etc. According to the characteristics of the DNA fingerprint diagram spectrum, a DNA fingerprint database is established to automatically compare, distinguish and analyze the DNA fingerprint spectrum and data of the identification biodiversity with the standard biodiversity to ensure the biodiversity to be identified. The present invention has the advantages of good repeatability, stabilization, reliability and high sensitivity, the automation of DNA molecular level identification analysis of the biodiversity is realized, and a powerful tool for accurate identification of the biodiversity is provided. The present invention provides a supporting system for establishing the DNA fingerprint database in a large scale and has important application value to medical treatment, medicine examination and departments of agriculture, forestry, graziery, customs, forensic physician, etc.

Description

Biological variety genome DNA fingerprint atlas

[technical field] the present invention relates to construction process and the Application of Fingerprint that a kind of biological variety comprises plant, animal, microorganism and human genome DNA's finger printing.

[background technology] biological variety comprises plant, animal, microorganism and the mankind's etc. evaluation and identification, mainly identifies according to methods such as its source, proterties, micro-and physics and chemistry at present, still lacks a kind of science, objective, identification of means fast.(amplified fragmentlength polymorphism is that Vos equals the technology that is used for the constructed dna fingerprint image that nineteen ninety-five develops on the basis of Zabeau AFLP) to amplified fragment length polymorphism DNA, belongs to third generation molecular genetic marker new technology.With first-generation molecular genetic marker-restriction fragment length polymorphism (restriction fragment lengthpolymorphism, RFLP) and s-generation molecular genetic marker-randomly amplified polymorphic DNA (randomamplification polymorphism DNA, RAPD) the dna fingerprint figure of Gou Jianing compares, this technology combines the reliability of RFLP and the high efficiency characteristics of RAPD, have good reproducibility, reliable and stable, rich polymorphism and highly sensitive advantage, showed that the AFLP technology is particularly suitable for using value and bright prospects that biological variety is identified.

[summary of the invention] identifies new way, the new technology of automatization in order to open up biological variety, is widely used in plant, animal, microorganism kind and legal medical expert and identifies, special proposition the present invention.

The present invention uses molecular genetic marker technique-amplified fragment length polymorphism DNA (AFLP) structure biological variety and comprises genome DNA fingerprint atlas such as people, plant, animal, microorganism, feature according to dna fingerprinting, set up corresponding mathematical model, adopt computer technology to set up the dna fingerprinting database, the support programs that design a calculating machine realize that biological variety dna molecular level discerns automatically.

Concrete technical scheme is as follows: screening and identification of organism standard substance (plant, animal, microorganism etc.), the extraction and purification biological variety genome DNA, design and the synthetic restriction endonuclease that contains, the dna double chain joint that promptly contains the synthetic of rare nickase such as EcoRI etc. and restriction enzyme sites such as frequent nickase such as MseI simultaneously, cut biological variety genome DNA with associating enzymes such as rare nickase and frequent nickase such as EcoRI/MseI, and be connected template DNA with the dna double chain joint that designs corresponding synthetic with preparation AFLP, sequence according to the DNA joint base of synthetic, design 3 ' end has 3 and selects the primer of base to make radiation 5 ' end mark, carry out pcr amplification, the PCR product is through denaturing polyacrylamide gel electrophoresis, obtain the radioautograph dna fingerprinting, machine image reading apparatus scanning as calculated, obtain dna fingerprint data and dna fingerprinting, use Database Systems to set up the dna fingerprint database, set up dna fingerprinting similarity factor mathematical model, design dna finger printing data processing software is also set up identification back-up environment automatically, treat identification of organism kind and standard biological kind dna fingerprinting and data and carry out automatic comparison and discriminatory analysis, clear and definite biological variety to be identified is standard substance why.

Obtaining of original image

The dna fingerprinting photo that produces via radioautograph among the present invention scanned by the computer picture scanner deposit in the disk.Because the picture quality that obtains is better, the resolving power and the signal to noise ratio of image are higher, therefore only need carry out simple image pretreatment, comprise image rectification, gray scale linear transformation, remove noise etc.The image of handling well is at last deposited in the raw image data storehouse.

The foundation of mathematical model

According to the feature of the dna fingerprinting that makes up, set up the similarity factor model between biological variety standard substance and the biological variety to be identified.The migration distance of supposing the dna fingerprinting band is D, and then according to the order and the position of dna fingerprinting band, the discrimination formula that two sample DNA finger printing bands of comparison are same strap is:

$\frac{|D_i-{D_i}^{\&prime;}|}{D_i}<T$

Wherein, T: acceptable coefficient of similarity threshold values;

D _i: the migration distance of certain biological variety standard model dna fingerprinting i band;

D _i: the migration distance of biological variety sample dna fingerprinting i band to be identified.

The dna fingerprinting coefficient of similarity (T) of two samples is understood that the band number that the dna fingerprint position of two samples is identical and the ratio of both total band numbers, is formulated as:

$\frac{{2n}_{\mathrm{1,2}}}{n_1+n_2}$

Wherein, n _1,2: the number of two biological variety sample DNA finger printing same strap;

n ₁: total band number of certain biological variety standard model dna fingerprinting;

n ₂: total band number of biological variety sample dna fingerprinting to be identified.

The identification of standard substance band number and position

Biological variety is identified must be based on standard model.By collecting some biological variety standard models, prepare its dna fingerprinting, form the dna fingerprinting database of standard substance.By image tagged and identification, calculate the position and the sum of its band, and they are deposited in the corresponding spectrum data storehouse.Contain several fingerprint bands of a spectrum of sample in each width of cloth finger printing image, the band that has quantity not wait again on each bands of a spectrum, the identification software that present method provides are found out the wherein position of each band one by one, and mark in addition.

The calculating of product band to be identified position, band number is the same with the mark and the identification of standard substance.

The automatic identification of product to be identified

The evaluation of biological variety is summed up as the calculating of product to be identified and standard substance coefficient of similarity size,, thereby can makes identifying the automatic identification of product by definition to the coefficient of similarity scope.

The computer program flow process is described

The whole procedure flow process is: at first scan biological variety genome DNA fingerprint atlas, they are saved in the spectrum data storehouse, be divided into three parts then collection of illustrative plates is handled:

(1) pre-treatment of collection of illustrative plates image.The image of handling well is saved in the spectrum data storehouse once more;

(2) parameter acquiring of collection of illustrative plates image.At first read in collection of illustrative plates file to be processed, to the image defining operation zone of reading in, the border of identification bands of a spectrum in specified zone, dependence program Automatic Logos go out band, simultaneously can also rely on manual mode to mark the band that program can't be discerned automatically, the influence of the error that produces when remedying automatic identification has related parameter to be saved in the spectrum data storehouse band of identification;

(3) biological variety genome DNA fingerprint atlas to be identified and parameter thereof are read in the identification of biological variety to be identified, compare the similarity of two kinds of calculated with mathematical model that standard substance and follow procedure set up then, show at last or print relevant result.

The present invention uses multi-disciplinary theory and technologies such as absorbing molecular biology, computer, phytology, pharmacognosy, microbiology, zoology, mathematics, uses the genome DNA fingerprint atlas that biological variety that the AFLP technique construction goes out to have important commercial value comprises people, plant, animal, microorganism; According to the feature of dna fingerprinting, set up corresponding mathematical model, adopt computer technology to set up the dna fingerprinting database, the support programs that design a calculating machine are developed the horizontal automatic recognition system of biological variety dna molecular.

The present invention has good reproducibility, reliable and stable, highly sensitive advantage, has realized the automatization of the horizontal identification and analysis of biological variety dna molecular.The accurate evaluation that comprises people, plant, animal, microorganism etc. for biological variety provides strong tool, also, significant application value is arranged in departments such as medical treatment, medicine inspection, agricultural, forestry, livestock industry, customs, legal medical experts for the foundation of the extensive dna fingerprint database system that provides support.This hi-tech system puts goods on the market and will create huge economic and social benefit.

[description of drawings]

Fig. 1: the agarose gel electrophoresis result of medicinal plant genomic dna.Wherein 1～6 be respectively mountain ginseng (fresh), genseng (fresh), genseng (dry root), Radix Panacis Quinquefolii (dry root), introduce a fine variety Radix Panacis Quinquefolii (dry root), pseudo-ginseng (dry root), M be λ DNA EcoRI+HindIII molecular weight standard.

Fig. 2: medicinal plant AFLP fingerprinting.Wherein 1～7 be respectively mountain ginseng (fresh), genseng (fresh), genseng (dry root), Radix Panacis Quinquefolii (dry root), introduce a fine variety Radix Panacis Quinquefolii (dry root), pseudo-ginseng (dry root), Arabidopis thaliana.

[embodiment]

Embodiment 1:(1) material genseng, Radix Panacis Quinquefolii, the fresh or dry root of introducing a fine variety Radix Panacis Quinquefolii and pseudo-ginseng originate in China Jilin Province, Wisconsin, USA, China Jilin Province, Yunnan Province respectively.

(2) separation of plant genome DNA is selected not have and is gone mouldy and fresh or dry root 75% ethanol disinfection of the genseng of damaging by worms, Radix Panacis Quinquefolii and pseudo-ginseng, scrape and abandon epidermis, get the 1g tissue, put in the little mortar, add liquid nitrogen grinding and become powder, it is transferred in the 50ml plastic centrifuge tube, add 6 * CTAB extracting solution (6%CTAB, the 100mmolL of 60 ℃ of insulations immediately ^-1Tris-HCl pH8.0,20mmolL ^-1EDTA, 1.4molL ^-1NaCl, 2% mercaptoethanol, 5%PVP) mixing, 60 ℃ of insulation 1h, and jolting frequently.Add isopyknic chloroform-primary isoamyl alcohol (24: 1) extracting after being cooled to room temperature, put upside down mixing gently, 14,000rmin ^-1Centrifugal 10min draws 10%CTAB/NaCl solution (10%CTAB, 0.7molL that supernatant liquor adds 65 ℃ of 1/10 volume preheatings ^-1NaCl), hatch 10min under the room temperature, add isopyknic chloroform-primary isoamyl alcohol (24: 1) and repeat extracting once, draw supernatant liquor and add isopyknic 1 * CTAB precipitation buffering liquid (1%CTAB, 50mmolL ^-1Tris-HCl pH8.0,10mmolL ^-1EDTA, 1% dredges basic ethanol), put upside down mixing gently, 60 ℃ of insulation 30min, 9000rmin ^-1Centrifugal 10min carefully removes supernatant liquor, and the back adds the high salt TE of 1ml solution (10mmolL ^-1Tris-HCl pH8.0,0.1mmolL ^-1EDTA pH8.0,1molL ^-1NaCl) dissolving CTAB-DNA sediment composite, the dehydrated alcohol of 2.5 times of volumes of adding is placed 1h under the room temperature, 9000r.min ^-1Centrifugal 10min, remove supernatant liquor after, respectively wash secondary with 65% and 85% ethanol respectively, abandon washing lotion, after the seasoning and be dissolved in TE and hold liquid (10mmolL ^-1Tris-HC pH8.0,0.1mmolL ^-1EDTA) among the 400uL.Add 2 μ L RNase (1mg.L ^-1), 37 ℃ of reaction 15min add isopyknic phenol-chloroform-primary isoamyl alcohol (25: 24: 1) and each extracting of chloroform-primary isoamyl alcohol (24: 1) respectively once, repeat dehydrated alcohol precipitation and ethanol and clean the step, and centrifugal collection DNA is resuspended in 400uL TE solution.

(3) the analytical sampling product DNA 10 μ L of the mensuration of DNA agarose gel electrophoresis and DNA concentration and purity and sample-loading buffer 2 μ L mixings are splined on 0.8% sepharose, with 1 * TBE electrophoretic buffer, 5v.cm ^-1Electrophoresis 30min, 0.5 μ g.mL ^-1After the dyeing of bromination second, observations and photograph under the ultraviolet lamp, as shown in Figure 1; Get a certain amount of sample DNA, dilute 100 times, measure A with ultraviolet spectrophotometer ₂₆₀And A ₂₈₀Value, and calculate A ₂₆₀/ A ₂₈₀Ratio.DNA purity is according to A ₂₆₀/ A ₂₈₀Ratio in judgement, the result is as shown in table 1.

Table 1: the medicinal plant DNA A that ultraviolet spectrophotometer records ₂₆₀, A ₂₈₀And A ₂₆₀/ A ₂₈₀

Sample A ₂₆₀A ₂₈₀A ₂₆₀/ A ₂₈₀

( x±s，n＝3) ( x±s，n＝3)

Mountain ginseng (fresh) 0.132 ± 0.004 0.075 ± 0.002 1.76

Genseng (fresh) 0.100 ± 0.003 0.052 ± 0.002 1.92

Genseng (dry root) 0.048 ± 0.002 0.027 ± 0.003 1.78

Radix Panacis Quinquefolii (dry root) 0.064 ± 0.004 0.037 ± 0.004 1.73

Introduce a fine variety Radix Panacis Quinquefolii (dry root) 0.045 ± 0.004 0.025 ± 0.003 1.80

Pseudo-ginseng (dry root) 0.050 ± 0.002 0.028 ± 0.001 1.79

(4) contain 5 * reaction buffer, 5 μ L among the EcoRI/MseI endonuclease reaction system 25 μ L of genomic dna, genseng, Radix Panacis Quinquefolii, introduce a fine variety the Radix Panacis Quinquefolii genome and be 250ng, contrast mouseearcress (Arabidopsis thaliana, Ara) DNA 250ng, EcoRI/MseI 2.5U behind 37 ℃ of reaction 2h, is hatched 15min with deactivation restriction enzyme activity for 70 ℃, centrifugal reaction tubes is collected reaction system, and places on ice.

(5) the above-mentioned EcoRI/MseI enzyme that is connected of EcoRI/MseI genomic DNA fragment and manual splice is cut in the after product, adds manual splice solution [EcoRI/MseI manual splice, ATP0.4mmolL ^-1, Tris-HCl (pH7.5) 10mmolL ^-1, Mg (Ac) ₂10mmolL ^-1, KAc 50mmolL ^-1] 24 μ L, T ₄Dna ligase 1U.Room temperature is mixing gently, and as increase the in advance template of reaction of AFLP, remaining connection product is in-20 ℃ of reaction 2h, and gets ligation product 10 μ L with 10 times of TE dilutions, mixing, and as increase the in advance template of reaction of AFLP, remaining connection product is in-20 ℃ of preservations.

(6) the AFLP PCR reaction response system 50 μ L that increase in advance contain above-mentioned dilution template 5 μ L, primer mixture (containing dNTPs) the 40 μ L that increase in advance, 10 * PCR reaction buffer [Tris-HCl (pH8.4) 200mmolL ^-1, MgCl ₂15mmolL ^-1, KCl 500mmolL ^-1], Taq archaeal dna polymerase 1U.Centrifugal mixing, and at 2～3 mineral oil of reaction system surface coverage.94 ℃ of 30S, 52 ℃ of 1min, 72 ℃ of 1min circulate 20 times.With template as next step selectivity AFLP amplified reaction.

(7) selectivity AFLP pcr amplification reaction mixed system 150 μ L contain EcoRI primer (E-AA) 5 μ L and (use γ ³²PdATP carries out 5 ' end mark), MseI primer (M-CAG) (containing dNTPs) 45 μ L; Mixed system 2 (Mix2) 100 μ L contain sterilized distilled water 79 μ L, 10 * PCR damping fluid, 20 μ L, TaqDNA polysaccharase 2.5U; Selectivity AFLP amplification reaction system 20 μ L contain Mixl 5 μ L, Mix2 10 μ L, the reaction product that increases in advance 5 μ L.Centrifugal mixing, 2～3 mineral oil of reaction system surface coverage.94 ℃ of 1min, 65 ℃ of 1min, 72 ℃ of 1min 30s follow every circulation primary and reduce by 1 ℃ of annealing temperature, circulate 9 times; Follow 94 ℃ of 30s again, 56 ℃ of 30s, 72 ℃ of 1min circulate 23 times.

(8) polyacrylamide gel electrophoresis and radioautograph selectivity AFLP amplified production add equal-volume (20 μ L) methane amide dyestuff (methane amide 98%, EDTA 10mmolL respectively ^-1, tetrabromophenol sulfonphthalein, dimethylbenzene cyanines), centrifugal mixing, 94 ℃ of heat denatured 3min place on ice immediately.Get sample-loading buffer 3 μ L in 6% polyacrylamide gel (20: 1 acrylamides: methylene diacrylamide, urea 7.5mmolL ^-1, 1 * TBE electrophoretic buffer), behind the permanent power prerunning of the 40W 20min, to get PCR product 10 μ L again and be splined in the gel pore, the permanent power electrophoresis of 40W migrates to gel 2/3 length up to the dimethylbenzene cyanines, stops electrophoresis, about 2h of time.With glass plate and gel separation, polyacrylamide gel is put the gel dryer oven dry then,, read under the sheet lamp and take a picture, as shown in Figure 2 through radioautograph.

Claims (2)

1. biological variety genome DNA fingerprint atlas, it is characterized in that: use molecular genetic marker technique-amplified fragment length polymorphism DNA (AFLP) structure biological variety and comprise genome DNA fingerprint atlas such as people, plant, animal, microorganism, concrete technical scheme is as follows:

Screening and identification of organism standard substance, the extraction and purification biological variety genome DNA, design and the synthetic restriction endonuclease that contains, the dna double chain joint that promptly contains the synthetic of rare nickase and frequent nickase restriction enzyme site simultaneously, cut biological variety genome DNA with rare nickase and frequent nickase associating enzyme, and be connected template DNA with the dna double chain joint that designs corresponding synthetic with preparation AFLP, sequence according to the DNA joint base of synthetic, design 3 ' end has 3 and selects the primer of base to make radiation 5 ' end mark, carry out pcr amplification, the PCR product is through denaturing polyacrylamide gel electrophoresis, obtain the radioautograph dna fingerprinting, dna fingerprint data and dna fingerprinting are obtained in machine image reading apparatus scanning as calculated.

2. the using method of the described finger printing of claim 1, it is characterized in that: according to the feature of dna fingerprinting, use Database Systems to set up the dna fingerprint database, set up dna fingerprinting similarity factor mathematical model, design dna finger printing data processing software is also set up identification back-up environment automatically, treat identification of organism kind and standard biological kind dna fingerprinting and data and carry out automatic comparison and discriminatory analysis, clear and definite biological variety to be identified.