| Objective:Silver staining is mainly used in the field of nucleic acid and protein staining.It has the advantages of high sensitivity,simple and effective.Therefore,silver staining methods has been widely used,and is applied to a variety of detection kits.In 1979,it was first applied to dyeing,and gel silver staining method also experienced constant optimization and development in decades.Specific mechanism is not clear,but the effects of silver staining nucleotide type and its sequence on silver staining remain unclear.This study used oligonucleotide as a model to explore the nucleic acid of silver staining.From the preliminary experiment,we noticed that when the silver staining similar oligonucleotide sequence chain staining intensity difference is very big.Isothermal amplification technology(rolling circle amplification,RCA)is one of the nucleic acid amplification techniques,compared with polymerase chain reaction(PCR),isothermal amplification characteristics is constant reaction temperature.RCA is one of the most commonly used methods of isothermal amplification,because the RCA has the advantages of simple,sensitive,easy to operate,so the RCA is widely used in such as DNA polymorphism and nucleic acid detection,miRNA detection.Through the use of antibodies and DNA enzyme,RCA also can be used in the detection of the proteins and small molecules.Galactose is a kind of important monosaccharides,galactosemia is an inherited metabolic disorder disease.Determination of galactose is often used in food detection and biological industrial production.The method of determination of D-galactose mass spectroscopy and chromatography need expensive equipment.This study combined with silver staining method and RCA isothermal amplification,to develop an equipment-independent visual detection method.EcoRI is a type of restriction endonuclease that recognizes specific sequences and is an essential tool for enzymes in biological experiments.However,EcoRI enzyme activity detection methods have long been cumbersome.This study is based on the principle of RCA reaction in RCA.The restriction endonuclease cleavage site was designed on the circular template,and the RCA product was observed by the rapid tube staining method to rapidly detect the EcoRI enzyme activity.Methods:In this study,alkaline silver staining was used to dye polyacrylamide gel after electrophoresis.We choose four kinds of degeneracies.The base types R,Y,M and K were studied and oligonucleotide chains were designed respectively.It was found that there were significant differences in different dyeing patterns of degenerate bases.To further study the rule of silver staining of nucleic acid,the sequence of deep silver staining(AG)was designed on RCA circular template to make RCA react and amplify later.The long tandem(AG)repeats of ssDNA were added.The acrylamide gel used in PAGE is added to the bottom of EP tube to make it mixed with RCA.The mixture is mixed and solidified by adding catalyst.After fixing,dyeing,rinsing and coloring steps,there is a long tandem(AG)repeat sequence.The color change of RCA products can be seen directly by naked eyes,and the dyeing efficiency is related to the content of RCA template and the reaction time of RCA.GalR was added to RCA system to detect galactose.GalR recognition sequence is designed on RCA template.When GalR was added to the system,the RCA reaction could not be carried out and the gel would not be dyed.When D-Gal exists in the reaction system,the GalR can be released from the template,allowing the RC.A reaction to proceed,and the gel is deeply stained.By this method,it is easy to implement in EP tube.D-Gal is detected by naked eye with complex instument and equipment.The EcoRI recognition sequence was designed on the RCA circular template,and EcoRI can specifically recognize and cleave the RCA template,and destroy the RCA template to prevent the reaction from occurring.The EcoRI enzyme activity can be detected by this principle combined with the rapidly detect tube staining method.Results:Silver staining of polyacrylamide gel electrophoresis showed that the efficiency of degenerate base types varied greatly.Experiments among the four degenerate bases selected,R and M were obviously stained,but Y and K were hardly stained.The order of staining efficiency from high to low is R(A/G)>M(A/C)>K(G/T)>Y(C/T),which indicates that the types of nucleotides have obvious effect on silver staining.The dyeing efficiency of base "T" is the lowest.The results showed that the oligonucleotide composed of continuous purine(A/G)was easy to dye.(AG)n was selected as the repeat sequence of silver staining.From the results of silver staining,it can be seen that the efficiency of silver staining of nucleic acid chains increases functionally with the increase of the number of(AG)repeats.In RCA circular template,it can be seen from the dyeing results that the products amplified by the template(CT)are obviously dyed,while the products amplified by the template(CT)are obviously dyed.The color of the product amplified by template(AG)was very light and similar to that of the blank control.The template(AG)is amplified by RCA.The detection sensitivity is increased 100 times(detection line is 1ng-l0pg).RCA can be effectively utilized when combined with repressor-RCA semi-quantitative to detection of D-Gal.The EcoRI recognition sequence was designed on the circular template of RCA,and cut by restriction endonuclease EcoRI.As the concentration of EcoRI in the system increased gradually,the RCA product reduced the rapid tube staining result,and the gray value of gel silver staining.The concentration relationship was linear and the enzyme activity of EcoRI was successfully detected.Conclusion:This study further explored the rule of silver staining of nucleic acid,and found that purine was easily stained by silver and used this characteristic to construct a new staining system.A method for visualized detection of D-Gal in EP tube was established.This method is simple to operate and cheap for nucleic acid and other reagents.Bioassay provides a new way of thinking.In addition,we introduced EcoRI on RCA circular template recognition sequence,succeeded in rapid detection of enzyme activity. |
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