CN101880660A

CN101880660A - DNA small fragment preparation template p1251 in DL2000 based on PCR (Polymerase Chain Reaction) amplification and preparation system thereof

Info

Publication number: CN101880660A
Application number: CN 201010197811
Authority: CN
Inventors: 叶春江; 叶春和; 谷劲松
Original assignee: University of Jinan
Current assignee: Jinan University; University of Jinan
Priority date: 2010-06-11
Filing date: 2010-06-11
Publication date: 2010-11-10

Abstract

The invention relates to a DNA small fragment preparation template p1251 in DL2000 based on PCR (Polymerase Chain Reaction) amplification. The map of the DNA small fragment preparation template p1251 contains 100bp, 250bp and 100bp recombinant DNA fragments separated by 4 EcoRI enzyme cutting sites, and the structures of the three recombinant DNA fragments are E100E250E100E. P1251 serves as the PCR amplification template, and proper sites are selected at both sides of the core sequence (100-250-100bp) of the template to be served as primer sequences to carry out the amplification to obtain nine PCR product compounds of the following types: 100/250/500-100/250/500; and after each compound is completely digested by EcoRI, obtained products are DNA fragments in a DNA marker DL2000.

Description

DNA small fragment preparation template p1251 and preparation system thereof among the DL2000 of PCR-based amplification

Technical field

The present invention relates to the innovation of electrophoresis consumptive material dna molecular amount standard fabrication method in molecular biology and the genetically engineered research field.Specifically, the present invention designs with a kind of proprietary pcr template, has set up the preparation system of the DL2000DNA marker small segment of brand-new PCR-based amplification method.

Background technology

Dna molecular amount standard is meant the mixture of one group of known dna molecular of molecular weight, different according to the size of its molecular weight and mobility behind the electrophoresis, on gel, form the gradient (ladder) that a dna fragmentation distributes, in order to the molecular weight of unknown DNA sample in the indication electrophoresis process.At present, mostly dna molecular amount standard is available from biotech firm; Owing to the ubiquity and the necessity of its application, in order to cut down expenses, a lot of laboratories are oneself preparation molecular weight standard commonly used all simultaneously.Dna molecular amount standard on the domestic market from initial based on imported product, be the appearance of many own products of advocating peace with homemade goods up till now, its mysterious veil is slowly decorporated.According to the related gordian technique (process) of dna molecular amount standard fabrication, its preparation method can be divided into digestion with restriction enzyme and two kinds of methods of pcr amplification; Wherein the source of the dna molecular of digestion with restriction enzyme can be divided into natural genomic dna and artificial constructed plasmid vector again; Pcr amplification can be divided into the amplification and the how segmental amplification of single slice again.

1. be equipped with dna molecular amount standard by the cutting of restriction endonuclease enzyme

Utilize the restriction enzyme digestion sites that exists in the dna molecular (natural or artificial the adding), adopt suitable restriction endonuclease that it is digested, thereby form one group of dna fragmentation that molecular weight is different, be used as dna molecular amount standard.According to being divided into two kinds: the extraordinary DNA (as genome) and the artificial constructed plasmid DNA of natural origin by the source of dna digestion.The digestion with restriction enzyme of the genomic dna of natural origin is by separating the genomic dna molecule in certain source, digest with one or more restriction enzymes then, thereby produce a series of dna fragmentation combination, the genomic dna (λ DNA) that present modal this type of dna molecular is the Lamda phage, dna molecular amount standard by its generation has λ DNA/HindIII, λ DNA/EcoRI+HindIII etc., also can be that certain has a plurality of common restriction enzyme site plasmids, as pBR322DNA/AluI marker and pBR322DNA/BsuRI (HaeIII) marker, but used restriction endonuclease all is of little use, thereby cost is higher.The digestion with restriction enzyme of artificial constructed carrier (plasmid) is by disposable needed various dna fragmentations to be cloned on the carrier of high copy number, change intestinal bacteria over to, by the separation and purification of fermentation culture and plasmid DNA, the suitable enzyme of process is cut and promptly can be obtained a large amount of target dna fragments.The advantage of this method is to obtain target DNA fragment by colibacillary fermenting and amplifying plasmid; its preparative-scale is easy to amplification, and (50 liters fermentation system is easy to set up; and pcr amplification generally can only carry out in less than the system of 500 microlitres); the dna fragmentation band band on gel that obtains is sharp keen, and process repeatability is high.There is not the bad shortcoming of pcr amplification reproducibility.But because DNA small segment molecular weight is little, during electrophoresis band with respect to the big fragment of high-molecular weight DNA very a little less than, so above-mentioned recombinant vectors is suitable for preparation DNA small segment.This is to utilize recombinant vectors to prepare the open defect that exists in the various DNA marker systems.The preparation of present various DNA small segments is mainly based on the method for pcr amplification.

2. prepare dna molecular amount standard by pcr amplification

Because the amplification ability of PCR (the Polymerase Chain Reaction) dna fragmentation that technology is powerful, while is owing to present Taq archaeal dna polymerase engineering strain is wide-spread, utilize PCR method to produce dna molecular amount standard at present and do not have technical problem, cost mainly is purchasing of the synthetic and dNTP of primer, other PCR reagent such as Taq archaeal dna polymerase, template DNA, reaction Buffer etc. all can make by oneself.Because production efficiency is low, concentration intensity height, thereby limited its large-scale preparation and application.

For a long time, molecular biology reagent and genetically engineered research field lack the dedicated carrier of the compound recombinant vectors design used as pcr template and the amplification of a kind of DL2000 of being used for DNA marker dna fragmentation, and preparation system correspondingly; Low to overcome regular-PCR amplification generation dna fragmentation production efficiency, defectives such as labour intensity height; Thereby can produce the DNA small segment among the dna molecular amount standard DL2000 fast, on a large scale.

Summary of the invention

Through secular laboratory study, the present invention has exactly satisfied the demand, design and recombination to construct as the template p1251 of pcr amplification, and set up the preparation system of DNA small segment among the corresponding D NAmarkerDL2000, exactly satisfied the demand; Its purpose is: the template p1251 that DNA small segment pcr amplification among a kind of DL2000 is provided.

Further purpose of the present invention is: a kind of system of utilizing above-mentioned template p1251 to prepare DNA small segment among the DL2000 by pcr amplification and enzyme cut bonded method is provided.

To achieve these goals, die for preparation plate p1251 provided by the invention is the pcr amplification template that the DNA small segment prepares usefulness among a kind of DNAmarker of being specifically designed to DL2000; Its collection of illustrative plates wherein contains 3 recombinant dna fragments (100bp, 250bp, 100bp) as shown in Figure 1, and all there are two EcoRI restriction enzyme sites at every bar segment two ends, and the structure of three recombinant dna fragments is: E100E250E100E.

The construction process of the pcr amplification template p1251 of DNA marker DL2000 comprises the steps: among the present invention

It with the arabidopsis thaliana genomic dna respectively template, utilize the method amplification of PCR to obtain following dna fragmentation 100E, E250E, E100bp, 5 ' the end of wherein 3 ' of 100E terminal, E100, and two ends of 250bp have the site of EcoRI restriction enzyme.Three kinds of dna fragmentations connect in the mode of a kind of " being linked in sequence " after the EcoRI enzyme is cut, and increase to connecting product with terminal primer PF, PR then, and screening obtains 100-250-100bp and connects product.Be needed overhang A ' (adenine nucleotide) among its terminal TA of interpolation clone simultaneously; Above-mentioned connection product TA is cloned among the cloning vector pGEM-T easy, promptly obtains design template p1251.

The preparation system of required DNA small segment is among the preparation DL2000:

With p1251 is template, selects appropriate site as primer sites in its two terminal sequence (100-250-100bp) both sides, and pcr amplification can obtain following combination product: 100/250/500-

-100/250/-500/ is totally nine kinds of PCR products, and after the EcoRI enzyme was cut, products therefrom was 100,250 or 500bp, all can be used as the small segment among the DL2000.The combination of PCR product can regulate 100,250, the relative populations ratio between three kinds of dna fragmentations of 500bp.

Pcr template carrier p1251 provided by the invention is the pcr amplification template that a kind of DL2000 of being specifically designed to small segment prepares usefulness; Select by primer sites flexibly, can specific form DNA amplification small segment 100,250 and 500bp with complex body; The PCR product that is obtained is cut through the enzyme of EcoRI can discharge the target dna fragment.

Beneficial effect of the present invention is:

Design a kind of pcr amplification template p1251, wherein contained 100bp, 250bp, the 100bp recombinant dna fragment separated by 4 EcoRI restriction enzyme sites; Can be with the DNA small segment fragment (cutting) among the form efficient production DL2000 of complex body with p1251 by the EcoRI enzyme for the pcr amplification template.Compare with traditional pcr amplification method to have and consume few (as dNTP, archaeal dna polymerase, primer, reaction buffer, experiment consumptive material etc.), efficient height (how segmental amplification simultaneously), advantage such as time saving and energy saving.

Description of drawings

Fig. 1 is the collection of illustrative plates of the recombinant PCR template p1251 of preparation dna molecular amount standard DL2000 small segment special use.

Concrete embodiment

With reference to accompanying drawing, p1251 provided by the invention is the pcr amplification template that the DNA small segment prepares usefulness among a kind of DL2000 of being specifically designed to; Wherein contain 100bp, 250bp, the 100bp recombinant dna fragment separated by 4 EcoRI restriction enzyme sites, the structure of three recombinant dna fragments is: E100E250E100E.

Its DL2000DNA small segment prepares system:

With p1251 is the pcr amplification template, selects appropriate site as primer sequence in its core sequence (100-250-100bp) both sides, and amplification can obtain the PCR product complex body of following type:

100/250/500-

-100/250/500, totally 9 kinds of PCR product complex bodys;

Every species complex is behind the EcoRI complete degestion, and products therefrom is the dna fragmentation among the DNA marker DL2000.

Claims

1. the PCR-based technology is used for the pcr amplification template p1251 that DL2000 DNA small segment prepares usefulness, it is characterized in that: wherein contain 100bp, 250bp, the 100bp recombinant dna fragment separated by 4 EcoRI restriction enzyme sites in its collection of illustrative plates, the structure of three recombinant dna fragments is: E100E250E100E.

2. PCR-based technology according to claim 1 prepares the system of DNA small segment among the DL2000 by template p1251, it is characterized in that: with recombinant vectors p1251 is pcr template, select appropriate site as primer sequence in its core sequence (100-250-100bp) both sides, amplification can obtain the PCR product complex body of following type:

100/250/500-

-100/250/500, totally 9 species complexs; Every species complex is behind the EcoRI complete degestion, and products therefrom is the dna fragmentation (100bp, 250bp, 500bp) among the DNA marker DL2000.