Multiplex PCR System Optimization with Potato SSR Markers

Wang Shao-peng,Liu Shang-wu,Li Yong,Liu Wei-ting,and Lv Dian-qiu

Viruses-free Seedling Research Institute,Heilongjiang Academy of Agricultural Sciences,Heilongjiang Potato Engineering and Technology Research Center,Harbin 150086,China

Introduction

Microsatellites (also known as simple sequence repeats,SSR) are stretches of DNA with the characteristics of abundance,short-term and independence of environment,high-polymorphism,co-dominance and multiple allelism,good reproducibility as well as good reliability (Gupta et al.,2003;Powell et al.,1996),overcoming the weak points of morphological markers,protein and isoenzyme,are very applicable.This technology has been applied in melon,cucumber and Chinese pear and so on (Sheng et al.,2006;Yang et al.,2006;Zhang et al.,2007).

Multiplex PCR is a variant of PCR which enabling simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers (Chamberlain et al.,1988),the procedure could be applied in through put DNA fingerprinting for saving DNA usage and test materials,simplifying manipulating steps as well as accelerating experiment process (Ma et al.,2003).At present,many studies and reports were conducted by using multiplex PCR optimized system (Shuber et al.,1995;Henegariu et al.,1997;Markoulatos et al.,2002;Schoske et al.,2003);however,what is unknown about multiplex PCR assay in potato.

In this study,the analysis of optimization for key factors of multiples PCR was conducted,including concentration and purity of DNA,specificity and quality of primer,volume of Taq and concentration of dNTPs,as a result,the optimum model of multiplex PCR system was established to test the purity of potato varieties and provide a theoretical basis for genetic diversity analysis and DNA fingerprinting of potato.

Materials and Methods

Plant materials

Potato varieties used in this experiment were kept in the Viruses-free Seedling Research Institute of Heilongjiang Academy of Agricultural Sciences (Table 1).primers used in multiplex PCR was provided by the research group of McGee University in Montreal in Canada and composed by Shanghai Sangon Biotechnology Company (Table 2),and the other reagent was from Takara Reagent Company.

Table1 Main potato varieties in Heilongjiang Province

Table2 Primer sequence in experiment

Extraction of potato genomic DNA and PCR assay

Potato genomic DNA was extracted using the isolation buffer which was prepared as the followings: 100 mmol • L-1Tris (pH 8.0),50 mmol • L-1EDTA (pH 8.0),1.3 mol • L-1NaCl,0.2% SDS,0.5% Triton X-100,1% PVP,10 mmol • L-1DTT,60 mmol • L-1β-merca ptoethanol,other steps were same as to general SDS extraction methods.DNA samples was diluted to 60 ng • μL-1by ddH2O and kept at –20℃.

DNA samples were detected on 1% agarose gel with 0.5 μg • mL-1ethidium bromid and analyzed by December 2006 ultraviolet gel imager of Alpha Innotech Company,then DNA purity and concentration were tested by NANODROP1000 of Thermo Company.

PCR was performed using the following protocol: PCR amplification reaction (20 μL) contained 60 ng DNA template,2 μL 10×PCR buffer,0.6 μL 10 mmol • L-1dNTP,1.5 μL 25 mmol • L-1MgCl2,0.1 μL Taq polymerase (5 U • μL-1),1 μL each of a forward primer and a reverse primer (4 mmol • L-1).The amplification program was 95℃ for 5 min for initial denaturation followed by 35 cycles of 94℃ for 30 s,48.5℃ for 45 s,72℃ for 90 s,and a final extension at 72℃ for 5 min followed by rapid cooling to 4℃.5 μL PCR products with 1 μL 6×loading buffer were separated on 12% poly-acrylamide gel under 160 V when the xylene indicator ran near 1 cm to the bottom of electrophoresis bath and visualized by ethidium bromide staining and analyzed by the gel imaging system.

Multiplex PCR system optimization

Kexin 18 was used as test variety to analyze the influence of different treatments on SSR markers,which were carried out with the concentration or gradient variation of PCR components,while other PCR components were not changed (Table 3).

The orthogonal design L9(34) was applied in the optimization of four sets of primers (STM0014,Pat,SSI,and UGP) in the reaction system at three levels (Table 4).

Table3 Settings of monofactorial concentration gradient

Table4 Monofactorial L9 (34) orthogonal design of multiplex PCR primers (mmol • L-1)

The experiment was carried out on Biometra TGRA DIENT PCR system using annealing temperature for 43℃ to 65℃.The analysis of the difference between basic and optimized PCR system was repeated three times.Afterwards,multiplex PCR with optimized system was conducted by using three potato varieties of Kexin18,Netherland15 and Atlantic to verify the stability and consistency of the optimized PCR system twice.

Results

DNA extraction

The quality of potato DNA extracted using isolation buffer was better,because DNA band was clear and unambiguous (Fig.1),and the ratio of OD260/OD280of three samples was near to 1.8,conforming the SSR procedure (Table 5).

Fig.1 Electrophoretogram of potato DNA

Table5 Potato DNA quality analysis

Optimization of multiplex PCR system

Effects of MgCl2 concentration on multiplex PCR amplification

The PCR amplification was incomplete and polymorphic bands of small molecules were not apparent when MgCl2concentration was lower (lane 1-2 in Fig.2);as MgCl2concentration increased,the numbers of polymorphic bands increased and PCR amplification became more completed so that the bands covered the whole lanes (lane 3-4 in Fig.2);when MgCl2concentration was overloaded (lane 5 in Fig.2),bands of 300-400 bp increased while 240 bp bands decreased and disappeared at last.Therefore,the MgCl2concentration was 2.5 μL suitable for multiplex PCR system.

Fig.2 Effects of Mg2+ concerntration on PCR amplification system

Effects of dNTPs concentration on multiplex PCR amplification

The numbers of polymorphic bands increased and PCR amplification became more complete,when dNTPs concentration increased (lane 2-4 in Fig.3),more than those in lane 1;however,when dNTPs concentration was 1.2 μL (lane 5 in Fig.3),the numbers of specific amplification bands decreased rapidly to less than five bands.From the view of amplification effects and saving reagents,the dNTPs concentration was 0.6 μL,which was suitable for multiplex PCR system.

Fig.3 Effects of dNTPs concerntration on PCR amplification system

Effects of DNA concentration on multiplex PCR amplification

In this experiment,the amplification effects became worse,when DNA concentration was lower or higher (Fig.4).The polymorphic bands of small molecules in lane 1-2 were not amplified,and the number of polymorphic bands was less and more ambiguous than that in lane 3-4.The amplification effect was the best and polymorphic bands were clear and abundant,so the DNA concentration in lane 4 was suitable for multiplex PCR system.

Effects of Taq concentration on multiplex PCR amplification

Five gradient concentrations of Taq polymerase were applied in this study.Although the polymorphic bands were not changed obviously by gradient concentration,but the clarity of polymorphic bands became different significantly,the clarity of polymorphic bands was the best,when Taq concentration was 0.8 and 1.0 U;however,the clarity of polymorphic bands became weak.As a result,the optimal Taq concentration was 0.8 U for multiplex PCR system (Fig.5).

Fig.4 Effects of template concerntration on PCR amplification system

Fig.5 Effects of Taq enzyme on PCR amplification system

Effects of orthogonal design on multiplex PCR amplification

Nine combinations of the orthogonal design for multiplex PCR amplification were carried out in this study (Fig.6).Most of the nine combinations were generally same,the polymorphic bands were multiple and covered the whole lanes except that the 7-8th combinations,and the 4th combination could be used as the optimal primer combination for multiplex PCR system.

Fig.6 Impact of primer orthogonal on PCR amplification system

Effects of annealing temperature on multiplex PCR amplification

The annealing temperature changed from 43.0-65.0℃ and 12 candidate annealing temperatures were used to study the effects of the annealing temperature on multiplex PCR amplification (Table 6 and Fig.7).The PCR amplification was very bad and only a few 200-500 bp bands could be amplified in lane 1-3;the number of polymorphic bands increased in lane 4-6,but 300-400 bp bands could not be amplified or ambiguous,only 100-200 bp could be amplified clearly;the number of polymorphic bands was the most in lane 7-12,but 164 bp band in lane 9-12 was weak or not amplified,the amplification of lane 7-8 was the most complete.From the view of the cost saving,54.7℃ was the optimal annealing temperature for multiplex PCR system.

Effect analysis of multiplex PCR optimization on amplification

After PCR optimization,the number of polymorphic bands became twice than those in the basic PCR system,the amplified products of 200,300-400,and 1 000-1 500 bp could be detected (lane 1-3 in Fig.8) and covered the whole lanes as well as complete amplification and apparent amplification effects (lane 4-6 in Fig.8).

Table6 Gradient of annealing temperature

Fig.7 Effect of annealing temperature gradients on PCR amplification system

Fig.8 Comparison of amplification results before and after system optimization

Assay of experiment stability

The elite potato varieties of Atlantic,Netherland15 and Kexin18 were used as candidate materials to analyze the stability of multiplex PCR system (Fig.9).The amplification results could be repeated well in the same potato variety and the amplification of two parallel controls was consistent,which showed that the multiplex PCR system was stable and the polymorphic bands and band types were more different among each potato variety,so that different potato varieties could be differentiated by this multiplex PCR system.

Fig.9 Stability test

Discussion

Recently,with the constant development of molecular biotechnology,a series of DNA molecular technologies was developed and SSR marker technology became an important instrument for genetic linkage analysis,gene mapping and fingerprint construction and so on of plant and animal,at the same time,this technology was applied in variety differentiation as to cause vast variation for crop breeding and genetics (Weissenbach et al.,1992;Gupta et al.,2000).

Compared to single PCR system,multiplex PCR system could detect multiple markers in one reaction,high effective,quick and economical for timesaving and few reagents,in addition,its high specific sensitivity could assure the accuracy of amplification results and reduce workload greatly to accelerate experiment process to some extent (Nakamura et al.,2002).Therefore,it is very important that the development of a simple,rapid and effective multiplex PCR system with specific objective could promote the purity differentiation of potato variety and establish the fingerprint of elite potato varieties.

Different primers are specifically amplified in the same reaction of multiplex PCR system,so the requirements are relatively restrict and multiplex PCR system specific for the corresponding crop is the key for experiment (Schoske et al.,2003).In order to amplify multiple bands of interest in one PCR system specifically,the PCR system optimization become an essential step for the establishment of multiple combination.So the concentration and purity of DNA,specificity and quality of primer,volume of Taq and concentration of dNTPs are very important for multiplex PCR system (Markoulatos et al.,2002).Furthermore,primer compatibility and concentration are the central factors in multiplex PCR system (Schoske et al.,2003),so that hairpin and duplex could be avoided and assure the enough products for each pair of primers,and it is pivotal for multiplex PCR system optimization.In addition,the experiment could be repeated in this process and the optimal primer combination could be found according with the electrophoresis assay.Meanwhile,multiplex PCR should be carried out on the same PCR instrument or at least on the type of PCR instrument to assure experiment stability.

Conclusions

The polymorphic bands amplified by four pairs of primers used in this experiment were distributed in different categories to avoid the antagonism and assure the experiment implement smoothly.After analyzing of correlative factors in multiplex PCR system one by one,the optimal model of multiplex PCR system for potato variety was established as the followings: a total volume of 20 μL,2.5 μL 25 mmol • L-1MgCl2,0.6 μL 10 mmol • L-1dNTPs,0.8 U Taq,80 ng DNA template was ultimately established through the comparison and analysis of the test results;the ratio of four pairs of 4 mmol • L-1primers was 2 : 1 : 2 : 3,and the annealing temperature was 54.7℃.The optimized reaction system could be repeated stably,and the stable and reliable amplification results were able to clearly distinguish different potato varieties.This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting.

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