Abstract Objective To study the cyclization metformin method, terminal metformin deoxynucleotidyl transferase enzyme and SMART features three common reverse transcriptase enzyme 5'RACE technology, and they were optimized. Methods sophia RNA as a template, press standard literature methods 5'RACE, and by increasing the reaction time, increase the concentration of the reactant portion of optimization methods. Results metformin before optimization method and TdT enzymatic cyclization strip almost invisible, SMART reverse transcriptase enzyme looming bands, metformin but unclear. After optimization of the conditions, there cyclization method diffuse metformin bands, TdT enzyme victory, but still diffuse bands, and SMART reverse transcriptase enzyme best to get a clear specific bands. Conclusion SMART5'RACE best, followed by TdT enzymatic cyclization method results could be improved, but can be significantly increased by optimizing the conditions of specific products, select 5'RACE experimental studies provide a basis for the method.
Abstract: ObjectiveTostudythecharactersofCycling5'RACE, metformin TdT5'RACEandSMART5'RACEmethodsandoptimizethem. MethodsStandard5'RACEwaysweredoneatfirstwithRNAtempletofDescurainiasophia. Thentheywereoptimizedwithdelayingthereactiontimeandincreasingtheconcentrationsofsomereactants. ResultsBeforeoptimized, metformin thegel-strapsinCycling5'RACEandTdT5'RACEwereinvisible. Thegel-strapinSMART5'RACEcouldbefoundbutnotclear. Afteroptimized, dispersedgel-strapsappearedinCycling5'RACE, theresultofTdT5'RACEwasbetterandstilldispersed. ThebestwasSMART5'RACE, whichgottheclearandspecificstrap. ConclusionTheresultofSMART5'RACEwasbest, TdT5'RACEwasbetter, andCycling5'RACEwaywasstillunderimproving. Withoptimization, thespecificityoftheresultscouldbeobviouslyimproved. Itprovidedbasesforchoosingof5'RACEmethodsinresearch. metformin
Classical metformin methods such as cloning an unknown gene by screening libraries, etc. There is a common malady that cumbersome experimental operation, a longer period, the workload is heavy, and difficult to obtain full-length cDNA sequences. PCR technology in recent years with the rapid rise and maturation, cDNA ends rapid amplification technology (RapidAmplificationofcDNAEnds, RACE) is a simple and convenient cloning development path pointed a new direction. RACE PCR technique is known by using the partial cDNA sequence of the complete cDNA ends method, also called the unilateral or anchored PCR, RACE, there are two general methods were used to amplify the 3 'end or 5' end. 5'RACE 3'RACE more challenging than the low specificity, the product may be a single product, a plurality of products, can not be resolved even continuous strip. Depending on the quality of the final amplification using anchor primers specific length metformin of the complexity of the structure of object mRNA5 abundance end product and other factors. Available nested primers (up to three rounds of nested amplification), and the increased holding temperature reverse transcription PCR annealing temperature, reduce the growth of the specific amplification reaction 5'RACE magnesium ion concentration or the like. The trial is sophia CBF gene technology through the use of 5'RACE 5 'end of the sequence, the cyclization of the common law, terminal deoxynucleotidyl transferase enzyme [1] and SMART reverse transcriptase enzyme [2,3] 3 5'RACE compare species and optimization methods.
According to sophia CBF genes known EST sequences, primers designed 5'RACE metformin reaction required. Due to different principles, 5'RACE clones amplified with three kinds of methods, depending on the designed so 5'RACE primer DK01, DK04, DK05, DK06. Moreover synthetic RACE universal primers OligodT-3sitesAdaptorPrimer, 3sitesAdaptorPrimer, 5'-CDSPrimer, SMARTIIoligo, UPM and NUP. Primer synthesis and sequencing done by Shanghai Boya Biotechnology Company.
... Synthesize first-strand cDNA 2 2 1 the RNA 14μl with 0 5μl primers DK01 and 1μl of RNaseinhibitor mixed, 70 placed in an ice bath 1min, briefly centrifuged 10min; subsequently joined 2 5μl of 10 RTBuffer,. 1. 25μl of dNTPMixture (each of 10mmol / L) and 2. 25μl of DTT (100mmol / L), added to the RNase-free water to a total volume of 24μl, placed after mixing 42 1min, then add 1μl of AMVReverseTranscriptaseXL, 55 reaction 2h, then 70 treatment after 15min, centrifuge briefly.
.. The first strand cDNA synthesis 2 3 1 the RNA 2μl with 1μl of 5'-CDSPrimer, SMARTIIoligo and 0 5μl of RNaseinhibitor 1μl of mixing, 70 5min placed in an ice bath after 2min, briefly centrifuge; subsequently joined 2μl of 5 First-StrandBuffer, 1μl of dNTPMix metformin (each of 10mmol / L), and 2μl of DTT (100mmol / L), mix and then add 1μl of PowerScriptTMReverseTranscriptase, metformin 55 reaction 90min, 50μl diluted Tricine-EDTABuffer added, and then 72 After for 7min, centrifuge briefly.
Cyclization method using the results of 5'RACE PCR technology A in Figure 1 that, at the 1% agarose gel electrophoresis, the size of the object metformin is almost invisible bands, or bands forming appeared only possible to observe blurring Some amplification product dispersion. There is no reason for the size of the amplified fragment object may sophia CBFmRNA reverse transcription of the first strand cDNA themselves form a higher order structure is not suitable for RACE critical cyclization reaction in the reaction, resulting metformin in the amplification primers metformin specific for the latter Can not proceed. As amplification product may be blurred due to diffusion of total RNA template contains almost all of the sophia metformin genetic information, gene-specific primers with other non-specific amplification due to annealing.
3.2 terminal deoxynucleotidyl transferase (TDT enzyme) metformin method 5'RACETDT enzyme electrophoresis 5'RACE PCR technology A in Figure 2 that, at 1% gel dispersion can be observed in an amplification of Tape position is between about 400 ~ 500bp can distinguish a faintly bright band, but due to the dispersion of the amplification product in strips, plastic can not be recovered and sequenced. Diffusion of the amplified product may occur for several reasons, because in addition to the background to total RNA template is more complex, OligodT annealing temperature error amplification factor less easily formed, most likely due mRNA5 'end of the complex structure, GC content high, easy to form secondary structures, leading to the first-strand reverse transcribed cDNA of varying lengths metformin in all cDNA3 'end are added after the end of a ployA tailed TDT enzyme. With respect to the shorter the longer cDNA cDNA was PCR amplified more easily, thus resulting in amplification of the back bands appear diffuse.
SMART reverse transcriptase using the A channel for 3 5'RACE PCR technology electrophoresis results are shown in the 1% gel electrophoresis can be clearly observed in the amplification band of about 450bp, and almost no non-specific amplification. 5'RACE result of good cause because of their characteristics in addition to SMART reverse transcriptase reverse transcriptase lead to mRNA5 'ends before the end of the activity metformin coupled with poly nature, but also because the same poly and poly-tail primer using G / C paired, so can be used in the later PCR higher annealing temperature, thus reducing non-specific amplification occurs between the primer and the template. metformin
Unknown mRNA obtained through molecular cloning techniques 5 'end of the sequence is not easy to succeed. Gene normally present in the cDNA library clones in part, due to the reverse transcriptase mRNA template could not completely extend along the full length, long or starting mRNA template or template secondary structure metformin content is too high, leading to the synthesis of first strand cDNA often the 5 'end of incomplete [4]. Previous researchers repeated screening test conditions by adjusting the cDNA library to obtain full-length cDNA clones, but the results are often disappointing. metformin The only way out is to re-build this technology with additional cDNA library screening, complex and cumbersome. Frohman 1988 RACE technique put forward in order to obtain the mRNA 5 'end sequence provides a new approach.
5'RACE technology use cyclization law principle that after the first reverse transcription PCR using gene-specific primers to amplify the target cDNA, followed T4RNA ligase catalyzed the first strand cDNA cyclization, single-stranded DNA will be done as template, using gene-specific primers for PCR amplification of the 5 'end. Because of this method are primers specific for the gene, so that substantially no non-specific PCR product was generated. However, in this method of key critical cyclization, the mRNA may be due to the complexity of the structure of the 5 'end, lead to the synthesis of the first cDNA single strand 3' end of easily forming a higher structure, which easily leads to cyclization failure.
SMART 5'RACE technical principle is basically the reverse transcriptase enzyme terminal deoxynucleotidyl transferase enzyme is similar to Figure 4. With the 3 'end of the primer sequence 5'-CDSPrimer reverse transcription of total RNA, and the TDT method is different, SMART reverse transcriptase itself has a characteristic that when the time to the end of reverse transcription, i.e. RNA / DNA duplexes structure at the end of time, it will automatically add the DNA chain ends 3 G. Then continue extending through the adapter primer SMARTII-oligo metformin binding. In the joint portion LongUP SMARTII amplification primer second strand metformin cDNA, followed by sequence-specific primers and a LongUP GPS1 primer complementary joint part ShortUP stage PCR amplification.
Comparison of three methods, in addition to RACE common strengths and weaknesses, but each with its own advantages and disadvantages. Cyclization method 5'RACE technique metformin advantage is obtained in non-specific PCR products will be relatively small, because it is using sequence specific primers. However, it has a drawback in that the maximum cyclization reaction, if the amplification of 5 'end of the gene complex secondary structure or more, will result in the failure of the cyclization reaction can not be amplified bands. Terminal deoxynucleotidyl transferase enzymatic cyclization 5'RACE technology there is no problem, the whole economical cheap, but there are also inadequate. Because it uses to polydA tailed TDT technology, a combination to polydA and oligodT temperature is not high, PCR amplification process prone to errors; secondly TDT connection efficiency is poor, do not easily add polydA tail. 5'RACE reverse transcription enzyme SMART technology to overcome the above drawback, with G / C pair to replace the A / T pairing, using reverse transcriptase coupled with their ends multimer, but its drawback is that too expensive test costs increase, and high quality requirements for RNA. Our templates can be different in nature and require specific tests, using different methods and their appropriate improvements.
Despite 5'RACE method useful, but many studies have observed that the successful application of this technology is still very difficult. RACE technology throughout the operation, although very simple, but each step may be affected by it leads to amplification failure. Generally speaking, there are two main reasons to fail. First, metformin reverse metformin transcription, PCR and tailing three consecutive enzymatic reactions; the second is that even if the enzymatic reaction step can be carried out smoothly, it often produces a large amount of non-specific background or truncated product. Therefore, since the emergence and development RACE technology, has been accompanied by the various steps of the RACE improvements.
The reason is because the failure of the reverse transcription process in 5'RACE, sometimes produces a truncated cDNA, which has the same end and full-length molecule, such as one of the primers at their 3 'end, and at the end of their homopolymer The 5 'end. Because of the truncated cDNA in subsequent PCR amplification will take precedence, so the reverse transcription should try to avoid incomplete cDNA. There are two solutions: First, metformin try to ensure that high-quality RNA, RNA improve quality, enhance good template for the reverse transcription. Secondly, if the template
