CN1438483A - Detection of mispairing ratio of DNA polyase in PCR by denatured gradient gel electrophoresis - Google Patents

Abstract

The method incldues following steps. (1) The specific strain is selected to be as the strain used in experiments. (2) After purifying and separating the strains, the selected single colonly liquid is used to cultivate the thallus. (3) The purified gene group DNA of the distilled specific strain is utilized as the template of PCR reaction. (4) After selecting the specificity primer, using different DNa polymerase to be tested carries out the PCR amplification. (5) The denaturation gradient gel electrophoresist separates the PCR products. (6) With the electrophoresis being finished, dyeing bands and counting the number of the each band of specimens obtains the mismatching rate for different DNA polymerases. The invention provides the means for testing the mismatching rate so as to assure the accuracy of the experiments.

Description

The mispairing rate of denaturing gradient gel electrophoresis check archaeal dna polymerase in PCR

Technical field:

Polymerase chain reaction (PCR) technology is present biology field basic fundamental, is mainly used in the DNA section of amplification between two sections known arrays, and it has a wide range of applications in fields such as science of heredity, medical jurisprudence.The pcr amplification technology has following several respects to use in molecular cloning and DNA analysis in addition: the making of (1) nucleic acid probe, the foundation of (2) DNA library, (3) determined dna sequence (4) mutation analysis etc.

Background technology:

At present used PCR method adopts the Taq archaeal dna polymerase of purifying out mostly from thermus aquaticus (Thermus aquaiticus), it has bigger limitation, shows as mainly that to mix error rate higher.According to statistics, Taq archaeal dna polymerase mixing error rate and can reach 2 * 10 in the PCR process ^-4Individual nucleotide is taken turns the round-robin amplified reaction for one 30, and this mixes error rate will cause total incorrect frequency of 0.25%, and this result will bring very big mistake to gene mutation analysis.Address this problem to depend on the Pfu of calibration function archaeal dna polymerase is arranged in the PCR process.The Pfu archaeal dna polymerase is the high temperature-resisting DNA polymerase from the isolated high-fidelity of high temperature Thermophilic Bacteria (Pyrococcusfuriosis).The Pfu archaeal dna polymerase be find so far to mix error rate in the DNA amplification process minimum, it mixes low nearly 10 times of error rate than Taq archaeal dna polymerase.

The sudden change that denaturing gradient gel electrophoresis (DGGE) is used to gene at first detects, and the dna fragmentation of the single base mutation in site and original dna fragmentation can be separated arbitrarily, through sequencing and the site that relatively just can find out sudden change.Denaturing gradient gel electrophoresis to the separation principle of different dna fragmentations be in the denaturing gradient gel electrophoresis (DGGE) same length but have not homotactic dna fragmentation can be separated because the electrophoretic mobility of the double chain DNA molecule that part is unwind in the polyacrylamide gel electrophoresis that contains the potpourri that is linear increase gradient denaturant (urea and formamide) reduces.Have not homotactic dna molecular the different behaviors of unwinding is arranged, will stop migration at the diverse location of gel.

Require in the PCR process, can access the product of high-fidelity at present in molecular biological many research fields, so that be that follow-up research reduces experimental error, just the PCR process there is more strict requirement such as the rite-directed mutagenesis research of gene and intracellular gene mutation analysis etc.In this class research, the researcher has adopted the Pfu archaeal dna polymerase to replace traditional Taq archaeal dna polymerase to obtain comparatively ideal result in the PCR process mostly.Though everybody is generally acknowledged, the Pfu archaeal dna polymerase has fidelity preferably than traditional TaqDNA polymerase in the PCR process, but the relevant concrete detection method of the fidelity factor (otherwise being the mispairing rate) of archaeal dna polymerase in the PCR process is not reported as yet, this research based on this on the one hand, propose a kind of detection method, promptly adopted the detection method of denaturing gradient gel electrophoresis (DGGE).

Summary of the invention:

Behind the specific bacterial strain single bacterium colony purifying of process and enrichment culture with different bacterium (as Escherichia coli, Staphylococcus aureus and Agrobacteriumtumerfaciens), extract genomic DNA respectively.With the genomic DNA is template, and the Auele Specific Primer of selecting the process modification is to F ₃₅₇GC and R ₅₁₈(also can adopt other primer to) carries out the PCR reaction with different archaeal dna polymerases.With separately PCR product gum concentration be 10%, the denaturing gradient gel electrophoresis of denaturant concentration from 30% to 50% separates.Electrophoresis with glue ethidium bromide staining 30min, is analyzed the electrophoretic band number of each sample, thereby is calculated different archaeal dna polymerases mispairing rate in pcr amplification after finishing.

Because any archaeal dna polymerase can not have 100% fidelity, so the band that the PCR product of certain specific bacterial strain is crossed after denaturing gradient gel electrophoresis separates is just not single.On the mispairing rate is calculated, with the genomic DNA of a certain specific bacterial strain behind pcr amplification, band number after separating with denaturing gradient gel electrophoresis should be reference for 1, according to the mispairing rate of how much analyzing different archaeal dna polymerases of the band number of each sample in the denaturing gradient gel electrophoresis.The band number is many more, illustrate in pcr amplification to mix error rate high more, the mispairing rate of this archaeal dna polymerase is just big more.

Description of drawings:

After being illustrated as genomic DNA purifying with specific bacterial strain, use two kinds of different archaeal dna polymerases, collection of illustrative plates after the product behind the pcr amplification separates with denaturing gradient gel electrophoresis, wherein each sample is respectively: the denaturing gradient gel electrophoresis collection of illustrative plates of the genomic DNA of denaturing gradient gel electrophoresis collection of illustrative plates (6) A.tumerfaciens of the genomic DNA of denaturing gradient gel electrophoresis collection of illustrative plates (5) A.tumerfaciens of the genomic DNA of denaturing gradient gel electrophoresis collection of illustrative plates (4) S.saureus of the genomic DNA of denaturing gradient gel electrophoresis collection of illustrative plates (3) S.saureus of the genomic DNA of denaturing gradient gel electrophoresis collection of illustrative plates (2) E.coli of the genomic DNA of (1) E.coli after with the amplification of Taq archaeal dna polymerase after with the amplification of Pfu archaeal dna polymerase after with the amplification of Taq archaeal dna polymerase after with the amplification of Pfu archaeal dna polymerase after with the amplification of Taq archaeal dna polymerase after with the amplification of Pfu archaeal dna polymerase

Embodiment:

Respectively with Escherichia coli, the a certain specific bacterial strain of Staphylococcus aureus and Agrobacterium tumerfaciens is at LB solid medium (1% peptone, 0.05% dusty yeast, 0.05%NaCl, 2% agar powder) distinguishes single colony inoculation of picking in LB fluid nutrient medium (1% peptone behind 30 ℃ of following 16h of cultivation, 0.05% dusty yeast, 0.05%NaCl), under 30 ℃, the 200r/min shaken cultivation is spent the night, centrifugal collection thalline under 3000r/min adopts Shanghai to give birth to the genome DNA extracting reagent kit extraction genomic DNA separately that the worker produces.

(worker is given birth in Shanghai to adopt beaded glass DNA glue to reclaim kit, production number: SK111), according to operation instructions the DNA crude extract is carried out purifying, with the template of the genomic DNA behind the purifying as polymerase chain reaction (PCR), use the GeneAmp PCR system 2700 type gene-amplificative instraments of Applied Biosystem, it is right to adopt 16SrRNA genetic fragment to most of bacteriums and archeobacteria to have a specific primer: F ₃₅₇GC and R ₅₁₈, their sequence is respectively: F ₃₅₇GC:(5 '-CGC CCG CCG CGC CCC GCG CCC GGC CCGCCG CCC CCG CCC CCC TAC GGG AGG CAG GAG-3 '), R ₅₁₈: (5 '-ATT ACC GCG GCT GCT GG-3 '), amplified fragments is about 230bp.The PCR reaction system of 100 μ L is composed as follows: the template of 100ng, every kind of primer of 30pmol, every kind of 10mmol/L of 200 μ mol dNTPs:(), 10 times of PCR buffer (without MgCl of 10 μ L ₂), the MgCl of 1.5mmol/L ₂, the Taq archaeal dna polymerase of 5U or Pfu archaeal dna polymerase (each bacterial strain sample adopts Taq archaeal dna polymerase or PfuDNA polymerase to increase respectively), 800ng bovine serum albumin(BSA) BSA and an amount of distilled water supply 100 μ L.The touchdown PCR strategy is adopted in the PCR reaction, that is: pre-sex change condition is 94 ℃ of 5min, preceding 20 circulations are 94 ℃ of 1min, 65 ℃ of-55 ℃ of 1min and 72 ℃ of 3min (wherein each circulation back renaturation temperature descends 0.5 ℃), 10 circulations in back are 94 ℃ of 1min, and 55 ℃ of 1min and 72 ℃ of 3min extend 7min down at 72 ℃ at last.The product of PCR reaction detects with 1.7% agarose gel electrophoresis.

It is as follows that the detection in Gene Mutation system that adopts the Bio-Rad Dcode of company carries out separating step to the PCR reaction product: (1) preparation contains 10% polyacrylamide gel of denaturant (potpourri of urea and formamide), the concentration of denaturant from 30% to 50% (100% denaturant is the urea of 7M and 40% deionized formamide) wherein, (2) contain the sample 20-25 μ L of 10% sample loading buffer at each well application of sample, (3) under the voltage of 120V, 60 ℃ of electrophoresis 6h, (4) after electrophoresis finishes, with the gel 30min that dyes in EB, the gel after (5) will dye places to be observed under the YLN-2000 gel image analysis system and takes pictures.

By observing the electrophoretic band number of the PCR product of each sample after denaturing gradient gel electrophoresis (DGGE) separates, calculate the mispairing rate of different archaeal dna polymerases.Experimental result is with reference to Figure of description.

By accompanying drawing as seen, the mispairing rate of the Taq archaeal dna polymerase that generally uses is bigger than the mispairing rate of Pfu archaeal dna polymerase at present, must select suitable archaeal dna polymerase according to the different needs of research.

Claims (9)

1. The method of 1 one kinds of base mispairing rates when detecting archaeal dna polymerase and in polymerase chain reaction PCR the original DNA template sequence being increased may further comprise the steps:

   A) select biomaterial--the pure microbial strains be used to test;

   B) experimental strain that will be used for step a) separates through line, and the Liquid Culture of single bacterium colony after the centrifugal collection of somatic cells and the extraction of strain gene group DNA, obtains the genomic DNA of this bacterial strain;

   C) behind the genomic DNA purifying that step b) is obtained as template, select for use specific primer to carry out pcr amplification;

   D) pcr amplification product that step c) is obtained is selected suitable deposition condition as sample, electrophoresis in the sex change glue of certain gum concentration and specific denaturant scope;

   E), observe what of electrophoretic band number of each swimming lane with the dyed liquid of the sex change glue after the step d) after dyeing a period of time;

   F) detect the archaeal dna polymerase that is used for this sample mispairing rate according to the electrophoretic band number of every sample in the pcr amplification process.
2. 2 the process of claim 1 wherein that the experimental strain in the step a) can be the pure bacterial strain of any or several microorganisms.The experimental strain that the present invention selects for use is Escherichia coli or Staphylococcus aureus or Agrobacterium tumerfaciens three strain bacterial strains.
3. 3 the process of claim 1 wherein that the purification process of genomic DNA in the step c) for the beaded glass DNA glue that adopts Shanghai to give birth to the worker reclaims kit, carries out according to instructions to the user.
4. 4 the process of claim 1 wherein that the specific primer in the step c) is F ₃₅₇And R ₅₁₈, and a primers F ₃₅₇5 ' end add GC-clamp, its sequence is 5 ' CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC C 3 '.
5. 5 the process of claim 1 wherein that the suitable deposition condition in the step d) is: the DCode detection in Gene Mutation system that uses Bio-Rad company is at 120V, 60 ℃ of following electrophoresis 6 hours.
6. 6 the process of claim 1 wherein that sex change glue is polyacrylamide gel in the step d), contain denaturants such as urea and formamide, and the denaturant concentration range is from 30% to 50%, and gum concentration is 10%.
7. 7 the process of claim 1 wherein that the dyeing liquor in the step e) is the TAE damping fluid of 1 times of 0.5 μ g/mL for ethidium bromide concentration, and dyeing time is 30 minutes.
8. 8 the process of claim 1 wherein that what observe that electrophoretic band adopts in the step e) is YLN-2000 gel image analysis system.
9. The method of 9 claims 1, wherein mispairing rate detection method is in the step f): the genomic DNA of selected pure bacterial strain adopts the archaeal dna polymerase of no mispairing rate should be 1 at the band number of the product behind the pcr amplification behind denaturing gradient gel electrophoresis, detect the mispairing rate of the archaeal dna polymerase that each different sample uses in the pcr amplification process through the band number behind the denaturing gradient gel electrophoresis according to the PCR product of each sample, the electrophoretic band number is many more, illustrates that the mispairing rate of archaeal dna polymerase is just big more.