Multiplex PCR System Optimization with Potato SSR Markers

September 2012

ScienceDirect

Vol. 19 No. 3 20-27

Journal of Northeast Agricultural University (English Edition)

Available online at www.sciencedirect.com

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

Abstract: Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. Concentration and gradient experiments for four components (enzyme, MgCl2, DNA template and dNTPs) in PCR system were used in the research with the concentration of the other component remained the same; 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; the temperature gradient selection was used to find out the optimum annealing temperature for the primer. The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL : 2.5 μL 25 mmol • L-1 MgCl2, 0.6 μL 10 mmol • L-1 dNTPs, 0.8 U Taq, 80 ng DNA template was ultimately established through the comparison and analysis of test results; the ratio of four pairs of 4 mmol • L-1 primers 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. . Key words: potato, SSR marker, multiplex PCR system, optimization CLC number:S532

Document code: A

Article ID: 1006-8104(2012)-03-0020-08

　Multiplex PCR is a variant of PCR which enabling

Introduction

simultaneous amplification of many targets of in-

Microsatellites (also known as simple sequence

primers (Chamberlain et al., 1988), the procedure

repeats, SSR) are stretches of DNA with the charac-

could be applied in through put DNA fingerprinting

teristics of abundance, short-term and independence

for saving DNA usage and test materials, simplifying

of environment, high-polymorphism, co-dominance

manipulating steps as well as accelerating experiment

and multiple allelism, good reproducibility as well

process (Ma et al., 2003). At present, many studies

as good reliability (Gupta et al., 2003; Powell et al.,

and reports were conducted by using multiplex PCR

1996), overcoming the weak points of morphological

optimized system (Shuber et al., 1995; Henegariu

markers, protein and isoenzyme, are very applicable.

et al., 1997; Markoulatos et al., 2002; Schoske et al.,

This technology has been applied in melon, cucumber

2003); however, what is unknown about multiplex

and Chinese pear and so on (Sheng et al., 2006; Yang

PCR assay in potato.

et al., 2006; Zhang et al., 2007).

　In this study, the analysis of optimization for key

terest in one reaction by using more than one pair of

Received 12 May 2011 Supported by the International Cooperation Project of Heilongjiang Science and Technology Department (WC05B08) Wang Shao-peng (1981-), male, Master, engaged in the research of identification of potato purity and molecular biology. E-mail: wangshaopeng 2010@126. com. * Corresponding author. Lv Dian-qiu, associate researcher, engaged in the research of potato cultivation and molecular biology. E-mail: smallpotatos@ 126. com. E-mail: [email protected]

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Wang Shao-peng et al. Multiplex PCR System Optimization with Potato SSR Markers

factors of multiples PCR was conducted, including

longjiang Academy of Agricultural Sciences (Table 1).

concentration and purity of DNA, specificity and

primers used in multiplex PCR was provided by the

quality of primer, volume of Taq and concentration of

research group of McGee University in Montreal

dNTPs, as a result, the optimum model of multiplex

in Canada and composed by Shanghai Sangon

PCR system was established to test the purity of potato

Biotechnology Company (Table 2), and the other

varieties and provide a theoretical basis for genetic

reagent was from Takara Reagent Company.

diversity analysis and DNA fingerprinting of potato. 　

Table 1　Main potato varieties in Heilongjiang Province

Materials and Methods

No.

Name of variety

Class

Plant materials

1

Atlantic

Breeder seed

Potato varieties used in this experiment were kept in

2

Netherland 15

Breeder seed

3

Kexin 18

Breeder seed

the Viruses-free Seedling Research Institute of HeiTable 2　Primer sequence in experiment Primer name

Sequence from 5' to 3'

Temperature (℃)

Annealing temperature (℃)

SSI-F

TCTCTTGACACGTGTCACTGAAAC

70

65

SSI-R

TCACCGATTACAGTAGGCAAGAGA

70

65

Patatin-F

CAACCAACAAGGTAAATGGTACC

66

61

Patatin-R

TGGTCTGGTGCATTAGAAAAAA

60

55

STM0014-F

CAGTCTTCAGCCCATAGG

56

51

STM0014-R

TAAACAATGGTAGACAAGACAAA

60

55

UGP-F

GAAACTGCTGCCGGTGC

56

51

UGP-R

TGGGGTTCCATCAAAC

48

43

pany. Extraction of potato genomic DNA and PCR

　PCR was performed using the following protocol:

assay

PCR amplification reaction (20 μL) contained 60 ng

Potato genomic DNA was extracted using the isolation

DNA template, 2 μL 10×PCR buffer, 0.6 μL 10 mmol• L-1

buffer which was prepared as the followings: 100

dNTP, 1.5 μL 25 mmol • L -1 MgCl 2 , 0.1 μL Taq

mmol • L-1 Tris (pH 8.0), 50 mmol • L-1 EDTA (pH 8.0),

polymerase (5 U • μL-1), 1 μL each of a forward primer

1.3 mol • L -1 NaCl, 0.2% SDS, 0.5% Triton X-100,

and a reverse primer (4 mmol • L-1 ). The amplification

1% PVP, 10 mmol • L-1 DTT, 60 mmol • L-1 β-merca

program was 95℃ for 5 min for initial denaturation

ptoethanol, other steps were same as to general SDS

followed by 35 cycles of 94℃ for 30 s, 48.5℃ for 45 s,

extraction methods.DNA samples was diluted to 60

72℃ for 90 s, and a final extension at 72℃ for 5 min

-1

ng • μL by ddH2O and kept at –20℃.

followed by rapid cooling to 4℃. 5 μL PCR products

　 DNA samples were detected on 1% agarose gel

with 1 μL 6×loading buffer were separated on 12%

-1

with 0.5 μg • mL ethidium bromid and analyzed by

poly-acrylamide gel under 160 V when the xylene

December 2006 ultraviolet gel imager of Alpha In-

indicator ran near 1 cm to the bottom of electro-

notech Company, then DNA purity and concentration

phoresis bath and visualized by ethidium bromide

were tested by NANODROP1000 of Thermo Com-

staining and analyzed by the gel imaging system. http: //publish.neau.edu.cn

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Journal of Northeast Agricultural University (English Edition)

Vol. 19　No. 3　2012

gradient variation of PCR components, while other Multiplex PCR system optimization

PCR components were not changed (Table 3).

Kexin 18 was used as test variety to analyze the

　The orthogonal design L9 (3 ) was applied in the opti-

influence of different treatments on SSR markers,

mization of four sets of primers (STM0014, Pat, SSI,

which were carried out with the concentration or

and UGP) in the reaction system at three levels (Table 4).

4

Table 3　Settings of monofactorial concentration gradient

Gradient

Factor

1

2

3

5

4

-1

25 mmol • L MgCl2 (μL)

0.5

1.5

2.5

3.5

4.5

10 mmol • L-1 dNTP (μL)

0.4

0.6

0.8

1.0

1.2

DNA (ng) Template

20

40

60

80

100

Taq polymerase (U)

0.4

0.6

0.8

1.0

1.2

Table 4　Monofactorial L9 (34) orthogonal design of multiplex PCR primers (mmol • L-1) No.

STM0014

SSI

Pat

UGP

1

0.1

0.1

0.2

0.3

2

0.1

0.2

0.3

0.2

3

0.1

0.3

0.1

0.1

4

0.2

0.1

0.2

0.3

5

0.2

0.2

0.3

0.2

6

0.2

0.3

0.1

0.1

7

0.3

0.1

0.2

0.3

8

0.3

0.2

0.3

0.2

9

0.3

0.3

0.1

0.1

　The experiment was carried out on Biometra TGRA

unambiguous (Fig. 1), and the ratio of OD260/OD280 of

DIENT PCR system using annealing temperature for

three samples was near to 1.8, conforming the SSR

43℃ to 65℃. The analysis of the difference between

procedure (Table 5).

basic and optimized PCR system was repeated three times. Afterwards, multiplex PCR with optimized sys-

1

tem was conducted by using three potato varieties of

2

3

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

Fig. 1　Electrophoretogram of potato DNA

buffer was better, because DNA band was clear and

1-3, DNA of potatoes.

E-mail: [email protected]

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Wang Shao-peng et al. Multiplex PCR System Optimization with Potato SSR Markers

amplification became more complete, when dNTPs Table 5　Potato DNA quality analysis

concentration increased (lane 2-4 in Fig. 3), more than

No.

OD260

OD280

OD260/OD280

Productivity (g • mL-1)

1

0.943

0.528

1.78

467

2

0.831

0.479

1.72

421

3

0.872

0.487

1.79

412

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.

Optimization of multiplex PCR system Effects of MgCl2 concentration on multiplex PCR

1

2

amplification

3

4

5

M 1 500 bp 1 000 bp

The PCR amplification was incomplete and polymorphic bands of small molecules were not apparent

500 bp 400 bp

when MgCl 2 concentration was lower (lane 1-2 in

300 bp

Fig. 2); as MgCl 2 concentration increased, the 200 bp

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);

100 bp

when MgCl2 concentration was overloaded (lane 5 in Fig. 2), bands of 300-400 bp increased while 240 bp bands decreased and disappeared at last. Therefore, the MgCl2 concentration was 2.5 μL suitable for multiplex PCR system.

1

2

3

4

5

Fig. 3　Effects of dNTPs concerntration on PCR amplification system 1, 0.4 μL; 2, 0.6 μL; 3, 0.8 μL; 4, 1.0 μL; 5, 1.2 μL; M, 100 bp DNA ladder Marker.

Effects of DNA concentration on multiplex PCR

M 1 500 bp 1 000 bp 500 bp 400 bp 300 bp

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

200 bp

polymorphic bands was less and more ambiguous than that in lane 3-4. The amplification effect was the

100 bp

Fig. 2　Effects of Mg2+ concerntration on PCR amplification system 1, 0.5 μL; 2, 1.5 μL; 3, 2.5 μL; 4, 3.5 μL; 5, 1.5 μL; M, 100 bp DNA ladder Marker.

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

Effects of dNTPs concentration on multiplex PCR

were not changed obviously by gradient concentration,

amplification

but the clarity of polymorphic bands became different

The numbers of polymorphic bands increased and PCR

significantly, the clarity of polymorphic bands was http: //publish.neau.edu.cn

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Journal of Northeast Agricultural University (English Edition)

Vol. 19　No. 3　2012

the best, when Taq concentration was 0.8 and 1.0 U;

as the optimal primer combination for multiplex PCR

however, the clarity of polymorphic bands became

system.

weak. As a result, the optimal Taq concentration was 0.8 U for multiplex PCR system (Fig. 5).

1

2 3

4

5 6

7

8

9 M 1 500 bp 1 000 bp

1

2

3

4

5

500 bp

M

400 bp

1 500 bp 1 000 bp

300 bp

500 bp 400 bp

200 bp

300 bp 200 bp

100 bp

Fig. 4　Effects of template concerntration on PCR amplification system 1, 20 ng; 2, 40 ng; 3, 60 ng; 4, 80 ng; 5, 100 ng; M, 100 bp DNA ladder Marker.

100 bp

Fig. 6　Impact of primer orthogonal on PCR amplification system 1-9, The same as in Table 3; M, 100 bp DNA ladder Marker.

Effects of annealing temperature on multiplex PCR amplification The annealing temperature changed from 43.0-65.0℃

1

2

3

4

5

and 12 candidate annealing temperatures were used

M 1 500 bp 1 000 bp 500 bp 400 bp 300 bp 200 bp

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

100 bp

clearly; the number of polymorphic bands was the most in lane 7-12, but 164 bp band in lane 9-12 was

Fig. 5　Effects of Taq enzyme on PCR amplification system

weak or not amplified, the amplification of lane 7-8

1, 0.4 U; 2, 0.6 U; 3, 0.8 U; 4, 1.0 U; 5, 1.2 U; M, 100 bp DNA ladder

was the most complete. From the view of the cost

Marker.

saving, 54.7℃ was the optimal annealing temperature for multiplex PCR system.

Effects of orthogonal design on multiplex PCR

Effect analysis of multiplex PCR optimization on

amplification

amplification

Nine combinations of the orthogonal design for

After PCR optimization, the number of polymorphic

multiplex PCR amplification were carried out in this

bands became twice than those in the basic PCR sys-

study (Fig. 6). Most of the nine combinations were

tem, the amplified products of 200, 300-400, and 1 000-

generally same, the polymorphic bands were multiple

1 500 bp could be detected (lane 1-3 in Fig. 8) and

and covered the whole lanes except that the 7-8th

covered the whole lanes as well as complete amplifica-

combinations, and the 4th combination could be used

tion and apparent amplification effects (lane 4-6 in Fig. 8).

E-mail: [email protected]

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Wang Shao-peng et al. Multiplex PCR System Optimization with Potato SSR Markers

Table 6　Gradient of annealing temperature Gradient

1

2

3

4

5

6

7

8

9

10

11

12

Temperature (℃)

43.0

43.5

45.0

47.4

50.0

52.4

54.7

57.0

59.5

61.9

63.7

65.0

M

1

2

3

4

5

6

7

8

9

10

11

12

1 500 bp 1 000 bp 500 bp 400 bp 300 bp 200 bp

100 bp

Fig. 7　Effect of annealing temperature gradients on PCR amplification system M, 100 bp DNA ladder Marker; 1-12, The same as in Table 6.

1

2

3 4

5

6

M

Assay of experiment stability

1 500 bp 1 000 bp

The elite potato varieties of Atlantic, Netherland15 and

500 bp 400 bp 300 bp

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

200 bp

same potato variety and the amplification of two parallel controls was consistent, which showed that the multiplex PCR system was stable and the poly-

100 bp

morphic bands and band types were more different

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

among each potato variety, so that different potato

1-3, Amplification results before system optimization; 4-6; Amplifica-

varieties could be differentiated by this multiplex PCR

tion results after reaction system optimization; M, 100 bp DNA ladder

system.

Marker.

M

1

2

3

4

5

6

7

8

9

10

11

12 1 500 bp 1 000 bp 500 bp 400 bp 300 bp 200 bp

100 bp

Fig. 9　Stability test 1-4, Amplification map of Kexin18, 1 and 2; 3 and 4 were two groups of repeated experiments Atlantic (5-8) and Favorita (9-12) were the same as group Kenxin18; M, 100 bp DNA ladder Marker. http: //publish.neau.edu.cn

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Journal of Northeast Agricultural University (English Edition)

Vol. 19　No. 3　2012

could be repeated in this process and the optimal

Discussion

primer combination could be found according with

Recently, with the constant development of molecular

should be carried out on the same PCR instrument

biotechnology, a series of DNA molecular technologies

or at least on the type of PCR instrument to assure

was developed and SSR marker technology became

experiment stability.

the electrophoresis assay. Meanwhile, multiplex PCR

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

Conclusions

was applied in variety differentiation as to cause vast

The polymorphic bands amplified by four pairs of

variation for crop breeding and genetics (Weissenbach

primers used in this experiment were distributed in

et al., 1992; Gupta et al., 2000).

different categories to avoid the antagonism and assure

　Compared to single PCR system, multiplex PCR

the experiment implement smoothly. After analyzing

system could detect multiple markers in one reaction,

of correlative factors in multiplex PCR system one by

high effective, quick and economical for time-

one, the optimal model of multiplex PCR system for

saving and few reagents, in addition, its high specific

potato variety was established as the followings: a total

sensitivity could assure the accuracy of amplification

volume of 20 μL, 2.5 μL 25 mmol • L-1 MgCl2, 0.6 μL

results and reduce workload greatly to accelerate

10 mmol • L-1 dNTPs, 0.8 U Taq, 80 ng DNA template

experiment process to some extent (Nakamura et al.,

was ultimately established through the comparison and

2002). Therefore, it is very important that the develop-

analysis of the test results; the ratio of four pairs of

ment of a simple, rapid and effective multiplex PCR

4 mmol • L-1 primers was 2 : 1 : 2 : 3, and the annealing

system with specific objective could promote the

temperature was 54.7℃. The optimized reaction

purity differentiation of potato variety and establish

system could be repeated stably, and the stable and

the fingerprint of elite potato varieties.

reliable amplification results were able to clearly dis-

　 Different primers are specifically amplified in

tinguish different potato varieties. This research built

the same reaction of multiplex PCR system, so the

the solid foundation for the further study of genetic

requirements are relatively restrict and multiplex PCR

diversity of potato germplasms and construction of

system specific for the corresponding crop is the key

DNA fingerprinting.

for experiment (Schoske et al., 2003). In order to amplify multiple bands of interest in one PCR system

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