CN109897886A - A kind of rice plumule Miho Dockyard NA template preparation method and PCR method for PCR amplification - Google Patents
A kind of rice plumule Miho Dockyard NA template preparation method and PCR method for PCR amplification Download PDFInfo
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- CN109897886A CN109897886A CN201910305392.5A CN201910305392A CN109897886A CN 109897886 A CN109897886 A CN 109897886A CN 201910305392 A CN201910305392 A CN 201910305392A CN 109897886 A CN109897886 A CN 109897886A
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Abstract
The present invention relates to field of biotechnology, more particularly to a kind of rice plumule Miho Dockyard NA template preparation method and PCR method for PCR amplification, present invention application alkali process Brown Rice is directly as PCR reaction template, its object is to found a kind of new method for quickly, effectively, easily carrying out DNA of plants amplification, this method is to remove the peel rice sample, 24 hours are cultivated under 37 DEG C of illumination to showing money or valuables one carries unintentionally, and obtain rice germ, for use；It takes in the investment reagent A of rice germ made from S1, boils 6-10min at 95-100 DEG C, add the reagent B of equivalent, stir evenly, obtain rice germ DNA profiling, using SSR standard primer, PCR amplification is carried out with the DNA profiling.The present invention is when carrying out kind of property verifying and auxiliary field breeding, with timeliness is strong, extraction step is simple, chemical reagent is less, smell is small and environmentally friendly technical effect.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of rice plumule Miho Dockyard NA template acquisition for PCR amplification
Method and PCR method.
Background technique
A kind of DNA molecular is used as since Batstein in 1980 etc. describes pvuii restriction fragment analysis (RFLP) for the first time
After label, with the development of molecular biology technology, the molecular marking technique of a variety of based on PCR is produced, these labelling techniques
The first step be that need to extract the genomic DNA of sample to be tested.People are frequently necessary to extract the DNA of plants of high quality, for constructing
The molecular biology experiments such as gene library, genome Sourthern analysis, digestion, clone and sequencing.According to the research pair of plant
As, research purpose and research cost etc. are different, it is also different to extract method applied by genomic DNA.Wherein, with crop molecule
The extensive use and popularization of assistant breeding, genotype identification are most important work, the extraction of genomic DNA be then it is most heavy,
One of most time-consuming work.At present using rice paddy seed as research material obtain DNA approach have 2 kinds: one is seed in perseverance
After sending out seedling in incubator, is converted to and extracts DNA with the method for blade；Another kind is directly to extract DNA with rice paddy seed, needs to grind
The various steps such as mill, centrifugation.The method for extracting paddy DNA has 5 kinds: 1) CTAB extraction method.The method is chiefly used in grass family plant
The extraction of object genomic DNA is that Doyle in 1987 is applied at first；2) isolation kit method.The method is mainly used in extraction
DNA high quality；3) SDS extraction method.The method is suitable for the extraction of various plants DNA.4) urea extraction method.The method is suitable for
The extraction of general plant and eukaryotic microorganisms DNA, nineteen ninety Dudler adopting said method at first.5) blade alkaline-heating method.This side
The object of method is rice, and template is used as after the direct soda boiling of blade.The advantage and disadvantage of these types of method are as follows:
Other methods use mortar, liquid nitrogen, centrifuge etc. in addition to alkaline-heating method, and extraction step is many and diverse, prepares chemistry examination
Agent huge number, extraction process pungent taste are big, and operating procedure is more.Although alkaline-heating method is very efficient, step is few, seed is had to
Experiment can just be carried out after hair seedling by 5-7 days, when carrying out kind of property verifying and auxiliary field breeding, test sample amount is more,
Just seem poor in timeliness.
Summary of the invention
The present invention creatively applies alkali process Brown Rice directly as PCR reaction template, and its object is to found one
Kind quickly, effectively, easily carries out the new method of paddy DNA amplification.
The first purpose of the invention is to provide one kind to be used for PCR amplification rice plumule Miho Dockyard NA template preparation method, including
Following steps:
S1, material processing
Rice sample is removed the peel, 24 hours are cultivated under 37 DEG C of illumination to showing money or valuables one carries unintentionally, obtain rice germ, for use；
S2, reagent prepare
Reagent A: the NaOH solution of concentration 3.5-4.5g/L；
The reagent B formula of every 500mL is as follows: 1mol/L pH8.0Tris-HCl5ml, 0.5mol/L EDTA1ml,
0.5mol/L HCl 100ml is added distilled water and is settled to 500ml；
The preparation of S3, rice germ DNA profiling
It takes in the investment reagent A of rice germ made from S1, boils 6-10min at 95-100 DEG C, add the reagent B of equivalent,
It stirs evenly, obtains rice germ DNA profiling.
A second object of the present invention is to provide a kind of method for carrying out PCR using above-mentioned rice plumule Miho Dockyard NA template, benefits
With SSR standard primer, PCR amplification is carried out with the DNA profiling that S3 in claim 1 is obtained.
Preferably, PCR reaction system are as follows: 1 × PCR buffer, 2.5mmol/L Mg2+, 0.25mmol/LdNTPs, 0.2 μ
Mol/L forward and reverse primer, 1.0U Taq archaeal dna polymerase, 20ng-40ngDNA template, 10 μ L of total volume；Response procedures are 95
DEG C 5min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, circulation 35 times, 72 DEG C of 5min obtain amplified production, 12 DEG C of preservations.
Compared with prior art, the method that a kind of rice germ of the invention is directly used in PCR amplification is imitated with following technology
Fruit:
Present invention alkali process crop seed to be measured is directly used as PCR reaction dna template, is carrying out kind of property verifying and auxiliary
When helping field breeding, in the case where test sample amount is big, need to carry out after seed sends out seedling compared to blade alkaline-heating method
Experiment, greatly shortens the time, and extraction step is simple, chemical reagent is less；It is extracted according to rice germ extraction DNA and kit
The result of DNA cloning effect compares, and the two effect is completely the same；Detect rice purity the results show that rice germ alkaline-heating method with
The purity probability that blade alkaline-heating method obtains is 94.3%, illustrates that the direct soda boiling of rice germ extracts DNA and can be used for hybrid rice kind
Sub- Purity；Likewise, rice germ alkaline-heating method is with blade alkaline-heating method detection validity the results show that the testing result of the two
Completely the same, the DNA for illustrating that rice germ alkaline-heating method extracts can also be used for rice seed Varieties identification.
Detailed description of the invention
Fig. 1 is that 4 rice germ blade alkaline-heating method of embodiment mentions DNA, the PCR for mentioning DNA with kit of comparative example 1 reacts amplification
As a result.
Fig. 2 is the paddy DNA purity detecting result that the blade alkaline-heating method of comparative example 2 extracts.
Fig. 3 is the paddy DNA purity detecting result that 4 rice germ alkaline-heating method of embodiment extracts.
Fig. 4 is the authenticity testing result that paddy DNA is extracted using the blade alkaline-heating method of comparative example 2.
Fig. 5 is the authenticity testing result that paddy DNA is extracted using the rice germ alkaline-heating method of embodiment 4.
Specific embodiment
With reference to the accompanying drawing and specific embodiment the present invention is described in detail, it is to be understood that guarantor of the invention
Shield range is not limited by the specific implementation.The method that actual conditions are not specified in the following example, usually according to routine
Conditional operation, the experimental material source being not specified be it is commercially available, due to not being related to inventive point, therefore its step is not retouched in detail
It states.
Embodiment 1
One kind being used for PCR amplification rice plumule Miho Dockyard NA template preparation method
S1, material processing
It takes 50g sample seeds brown rice machine to handle, rice germ is placed in germination box and is germinateed, germination box is put into 37 DEG C of light
It is stand-by to showing money or valuables one carries unintentionally according to 24 hours are cultivated in incubator；
S2, reagent prepare
Reagent A: the NaOH solution of concentration 4g/L；
The reagent B formula of every 500mL is as follows: 1mol/L Tris-HCl (PH8.0) 5ml, 0.5mol/LEDTA 1ml,
0.5mol/L HCl 100ml is added distilled water and is settled to 500ml；
The preparation of S3, rice germ DNA profiling
It takes the investment of rice germ made from S1 to fill in the container of reagent A, 8min is boiled at 100 DEG C, take out container and to appearance
The reagent B of equivalent is added in device, stirs evenly, obtains rice germ DNA profiling.
Embodiment 2
Essentially identical with the operation of embodiment 1, difference is, reagent A in S2: the NaOH solution of concentration 3.5g/L；In S3,
6min is boiled at 95 DEG C.
Embodiment 3
Essentially identical with the operation of embodiment 1, difference is, reagent A in S2: the NaOH solution of concentration 4.5g/L；In S3,
8min is boiled at 97 DEG C.
Through detecting, in embodiment 1-3, the DNA of embodiment 1 obtains extracting rate highest, is 100%, determines that embodiment 1 is optimal side
Case.
Embodiment 4
A method of PCR is carried out using rice plumule Miho Dockyard NA template
The rice plumule Miho Dockyard NA template for selecting embodiment 1 to extract, utilizes SSR standard primer RM7120 (SN/T3402-
2012, synthesized by the raw work in Shanghai), forward primer TGCCCAAAATATATGAAACC, as shown in SEQ ID NO.1, reverse primer
TTTTCTTGTTGAATGGGAAC, as shown in SEQ IDNO.2, reaction system are as follows: 1 × PCR buffer, 2.5mmol/L Mg2+,
0.25mmol/L dNTPs, 0.2 μm of ol/L forward and reverse primer, 1.0U Taq archaeal dna polymerase, 20ng-40ng sample DNA, always
10 μ L of volume；Response procedures are 95 DEG C of 5min, and 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, circulation 35 times, 72 DEG C of 5min are expanded
Increase production object, 12 DEG C of preservations.
Comparative example 1
The DNA and PCR amplification of RNA isolation kit extraction rice sample
S1, it takes 50g sample to be placed in mortar, liquid nitrogen is added by sample grind into powder, weighs what 70mg-100mg was ground
Sample is transferred in the Eppendorf pipe of 2.0ml；
S2, view quantity of material number, the warmed-up Buffer A of 600ul is added, after mixing gently, 65 DEG C of water-baths heat preservations
10min:
S3,600ul phenol/chloroform is added in Eppendorf pipe pipe, turns upside down and mixes well, extract 2min；
S4,12000 × g are centrifuged 5 minutes, and Aspirate supernatant is into a new Eppendorf pipe:
S5, addition and the isometric Buffer B of supernatant, mix, after room temperature 10min, 12000 × g centrifugation
5min removes supernatant, retains precipitating:
S6,60ulBuffer C is added in precipitating, is mixed well (very after 37 DEG C (room temperature) placement 2min with pipette tips
It is important) after, precipitated in 37 DEG C of dissolutions, 300ulBufferD be added after five minutes, turns upside down after mixing well 10 times, take out from
Centrifugal column is placed on the casing of a 2ml, solution is added in centrifugal column by stem, places 2min；
S7, centrifugal column and 2ml casing one are reinstated to 8000 × g centrifugation, after 30 seconds, discards solution in 2m1 casing, is being centrifuged
200u1Wash Buffer I is added in column, 8000 × g centrifugation after 30 seconds, discards solution；
S8,200u1Wash Buffer I is added in centrifugal column, 8000 × g centrifugation after 30 seconds, discards solution；
S9, add 200u1Wash buffer II in centrifugal column, 8000 × g centrifugation after 30 seconds, discards solution；
S10,200u1Wash buffer II is added in centrifugal column, 8000 × g centrifugation after 30 seconds, discards solution；11,
12000 × g is centrifuged 30 seconds, to remove traces of residual solution in centrifugal column；It is placed at room temperature for 10min；
S12, centrifugal column is placed in a new 1.5m1 centrifuge tube (asking white standby), it is careful in centrifugal column bottom center
After addition 130u1Elution Buffer, 37 DEG C of placement 10min, 12000 × g is centrifuged 1 minute；13.-20 DEG C of preservation.
S13, PCR amplification
Select SSR standard primer RM7120 (SN/T3402-2012 is synthesized by the raw work in Shanghai), forward primer
TGCCCAAAATATATGAAACC, as shown in SEQ ID NO.1, reverse primer TTTTCTTGTTGAATGGGAAC SEQ ID
Shown in NO.2, reaction system are as follows: 1 × PCR buffer, 2.5mmol/L Mg2+, 0.25mmol/L dNTPs, 0.2 μm of ol/L just,
Reverse primer, 1.0U Taq DNA polymerase, 20ng-40ng sample DNA, 10 μ L of total volume；Response procedures are 95 DEG C of 5min, 94
DEG C 30s, 55 DEG C of 30s, 72 DEG C of 40s, circulation 35 times, 72 DEG C of 5min obtain amplified production, 12 DEG C of preservations.
Comparative example 2
Blade alkaline-heating method extracts paddy DNA template and PCR amplification
S1, material processing
It germinates in germination box after taking the seed of 50g to be sterilized with hypochlorous acid, germination box is put into 37 DEG C of illumination box
Reach 2-3cm (tri-leaf period) for use to seedling length within culture 7 days, guarantees to be not less than 200 single plants.
S2, reagent prepare
Reagent A: reagent A: the NaOH solution of concentration 4g/L；
The reagent B formula of every 500mL is as follows: 1mol/L Tris-HCl (PH8.0) 5ml, 0.5mol/LEDTA 1ml,
0.5mol/L HCl 100ml is added distilled water and is settled to 500ml；
The preparation of S3, template DNA
Take rice leaf with tweezers it is tortuous after be put into the centrifuge tube for fill reagent A and boil 5min in boiling water, take out centrifugation
The reagent B of equivalent is added in Guan Bingxiang centrifuge tube, is stirred evenly with liquid-transfering gun, draws 2.0 μ l and is directly used in as DNA profiling
PCR reaction.
S4, PCR amplification
Select SSR primer RM7120 ((SN/T3402-2012 is synthesized by the raw work in Shanghai)), forward primer such as SEQ ID
Shown in NO.1, shown in reverse primer SEQ ID NO.2, reaction system are as follows: 1 × PCR buffer, 2.5mmol/L Mg2+,
0.25mmol/L dNTPs, 0.2 μm of ol/L forward and reverse primer, 1.0UTaq archaeal dna polymerase, 20ng-40ng sample DNA are overall
10 μ L of product；Response procedures are 95 DEG C of 5min, and 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, circulation 35 times, 72 DEG C of 5min are expanded
Product, 12 DEG C of preservations.
It is detected using native polyacrylamide gel electrophoresis
It takes 10 μ L embodiments 4, comparative example 1 and comparative example 2 to expand obtained product respectively, isometric sample-adding buffering is added
Liquid, runs denaturation program in PCR instrument after mixing well, 95 DEG C of denaturation 5min, 4 DEG C of cooling 10min or more, non denatured 6%
Constant pressure prerunning (100V constant pressure, 10~30min) on polyacrylamide gel, then constant pressure electrophoresis (200~250V constant pressure, 1~
2h), gel immerses in fixer, and being placed in the fixed 5min of shake on shaking table, (amount of fixer can be according to offset plate quantity and big ditty
It is whole, be subject to and do not cross glue surface), carries out that short rinse is primary with ddH2O, dyeing 10min, use are shaken in the dyeing liquor newly prepared
DdH2O (0.1% sodium thiosulfate containing 0.2% volume) short rinse, the time is no more than 10s, in the developer solution newly prepared
It shakes until show clearly band, 5min is fixed in fixer, result is directly recorded on film illuminator or with digital
Camera photograph.
Experimental result
1, rice rice germ mentions compared with DNA mentions the amplification of DNA with kit
The amplification of embodiment 4 and comparative example 1 is as shown in Figure 1, wherein 1,2 DNA extracted for rice germ, 3,4 be examination
The DNA that agent box extracts, can be seen from the chart, the DNA that both methods is extracted does not have difference, and band clearly becomes clear, no hangover,
Therefore the alkali process of rice germ is used directly for the template of PCR extension.
2, rice germ extracts application of the DNA in rice purity
Whether there is Parent complementation banding pattern according to sample single plant, determine whether it is true cenospecies, the benefit of comparative example 2
It is that template carries out Amplification Analysis to above-mentioned single plant with the DNA that blade alkaline-heating method extracts, 192 single plants of coamplification detect 14 plants of lifes
Object mixes (Fig. 2), wherein 14 plants maternal, 0 plant of male parent, purity probability is 92.7%；Embodiment 4 is subtracted using rice germ boils extraction
DNA be that template carries out Amplification Analysis to above-mentioned single plant, 192 single plants of coamplification detect 11 plants of biology hybrids (Fig. 3),
Middle maternal 11 plants, 0 plant of male parent, purity probability is 94.3%, not up to standard, both sides that are considered as purity according to total hybrid strain number > 4%
The testing result that method extracts DNA is completely the same, and the direct soda boiling of the rice germ of explanation extracts DNA and is fully available for hybrid rice seeds
Purity.
3, rice germ extracts application of the DNA in rice authenticity
According to the similarities and differences of sample room electrophoretic band, the authenticity by sample product is determined.For trying 3 kind A, B, C (A two
The examination of excellent 58 First Year area, B are that second year area tries, and C is third year production experiment) between comparison, each kind takes two lists
Strain is extracted and is expanded using the blade alkaline-heating method of comparative example 2, and augmentation detection is the results show that without Site discrepancy between sample A, B, C
(Fig. 4) determines that sample A, B, C are identical or extremely approximate kind, extracts and expand using the rice germ alkaline-heating method of embodiment 4, inspection
Survey determines that sample A, B, C are identical or extremely approximate kind the results show that without Site discrepancy (Fig. 5) between sample A, B, C, both
The DNA testing result that method is extracted is completely the same, and it is true to illustrate that the DNA of rice germ alkaline-heating method extraction is fully available for rice seed kind
Reality identification.
Above is only one embodiment of the present of invention, but the embodiment of the present invention is not limited only to this, any this field
Can think of variation should all fall within the scope and spirit of the invention.
Sequence table
<110>Paddy Rice Inst., Anhui Agriculture Science Academy
<120>a kind of method that rice germ is directly used in PCR amplification
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
tgcccaaaat atatgaaacc 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
ttttcttgtt gaatgggaac 20
Claims (3)
1. a kind of rice plumule Miho Dockyard NA template preparation method for PCR amplification, which comprises the steps of:
S1, material processing
Rice sample is removed the peel, is cultivated under 37 DEG C of illumination to showing money or valuables one carries unintentionally, obtains rice germ, for use；
S2, reagent prepare
Reagent A: the NaOH solution of concentration 3.5-4.5g/L；
Reagent B, and the reagent B formula of every 500mL is as follows: 1mol/L pH8.0Tris-HCl5ml, 0.5mol/L EDTA 1ml,
0.5mol/L HCl 100ml is added distilled water and is settled to 500ml；
The preparation of S3, rice germ DNA profiling
It takes in the investment reagent A of rice germ made from S1, boils 6-10min at 95-100 DEG C, add the reagent B of equivalent, stir
Uniformly, rice germ DNA profiling is obtained.
2. a kind of method for carrying out PCR using rice plumule Miho Dockyard NA template described in claim 1, which is characterized in that
Using SSR standard primer, PCR amplification is carried out with the rice germ DNA profiling.
3. the method according to claim 2 for carrying out PCR with rice plumule Miho Dockyard NA template, which is characterized in that PCR reaction
System are as follows: 1 × PCR buffer, 2.5mmol/L Mg2+, 0.25mmol/L dNTPs, 0.2 μm of ol/L forward and reverse primer, 1.0U
Taq archaeal dna polymerase, 20ng-40ngDNA template, 10 μ L of total volume；Response procedures are as follows: 95 DEG C of 5min, 94 DEG C of 30s, 55 DEG C
30s, 72 DEG C of 40s, circulation 35 times, 72 DEG C of 5min obtain amplified production, 12 DEG C of preservations.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111455025A (en) * | 2020-04-29 | 2020-07-28 | 安徽省农业科学院水稻研究所 | Rapid soybean DNA obtaining method for PCR amplification and PCR amplification method |
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