Single universal primer multiplex ligation-dependent probe amplification with sequencing gel electrophoresis analysis

Accepted Manuscript Single Universal Primer Multiplex Ligation-dependent Probe Amplification with sequencing gel electrophoresis analysis Ying Shang, Pengyu Zhu, Wentao Xu, Tianxiao Guo, Wenying Tian, Yunbo Luo, Kunlun Huang PII: DOI: Reference:

S0003-2697(13)00442-9 http://dx.doi.org/10.1016/j.ab.2013.09.012 YABIO 11493

To appear in:

Analytical Biochemistry

Received Date: Revised Date: Accepted Date:

27 July 2013 6 September 2013 9 September 2013

Please cite this article as: Y. Shang, P. Zhu, W. Xu, T. Guo, W. Tian, Y. Luo, K. Huang, Single Universal Primer Multiplex Ligation-dependent Probe Amplification with sequencing gel electrophoresis analysis, Analytical Biochemistry (2013), doi: http://dx.doi.org/10.1016/j.ab.2013.09.012

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Single Universal Primer Multiplex Ligation-dependent

2

Probe Amplification with sequencing gel electrophoresis

3

analysis

4

Ying Shang1**, Pengyu Zhu1**, Wentao Xu1,2*, Tianxiao Guo2, Wenying Tian2, Yunbo Luo1,2,

5

Kunlun Huang1,2,*

6

1Laboratory of Food Safety, College of Food Science and Nutritional Engineering, China

7

Agricultural University, Beijing 100083, China

8

2The Supervision, Inspection & Testing Center of Genetically Modified Food Safety, Ministry of

9

Agriculture, Beijing 100083, China

10

**These two authors contributed equally

11

*Corresponding author: Tel.: 86-10-6273-8793. Fax: 86-10-6273-7786.

12

E-mail address: [email protected] (K.L. Huang) and [email protected] (W.T. Xu)

13

Running title: SUP-MLPA with sequencing electrophoresis analysis

14

Number of figures: 4

15

Number of tables: 3

16

Figure 1 in color

17

The appropriate subject category: DNA Recombinant Techniques and Nucleic Acids.

1

18

Abstract

19

In this study, a novel single universal primer multiplex ligation-dependent probe

20

amplification (SUP-MLPA) technique that used only one universal primer to perform

21

multiplex PCR was developed. Two reversely complementary common sequences

22

were designed on the 5’ or 3’ end of the ligation probes, which allowed the ligation

23

products to be amplified through only a single universal primer (SUP). SUP-MLPA

24

products were analyzed on sequencing gel electrophoresis with the extraordinary

25

resolution. This method avoided the high expenses associated with capillary

26

electrophoresis, which was the commonly detection instrument. In comparison to

27

conventional multiplex PCR, which suffers from low sensitivity, non-specificity and

28

amplification disparity, SUP-MLPA had higher specificity and sensitivity, and low

29

detection limit of 0.1 ng for detecting single crop species when screening the presence

30

of genetically modified crops. We also studied the effect of different length of stuffer

31

sequences on the probes for the first time. Through comparing the results of qPCR,

32

the ligation probes (LPs) with different stuffer sequences did not affect the ligation

33

efficiency, which further increased the multiplicity of this assay. The improved

34

SUP-MLPA and sequencing gel electrophoresis method will be useful for food and

35

animal feed identification, bacterial detection and verification of genetic modification

36

status of crops.

37

Keywords: single universal primer, multiplex ligation-dependent probe amplification,

38

sequencing gel electrophoresis, genetically modified crops

2

39

INTRODUCTION

40

Rapid and highly sensitive detection of DNA is critical in genotyping of

41

individuals, food and feed identification, infectious diseases, environmental

42

monitoring, etc [1]. With the advent of modern analytical technologies, many

43

researchers and laboratories have taken advantage of new opportunities for nucleic

44

acid detection. However, due to the increased number of molecular targets to be

45

analyzed, assay miniaturization and cost-efficiency become necessary. Multiplex PCR

46

is a variation of conventional PCR that simultaneously amplifies multiple targets in

47

only one reaction [2]. This approach can potentially lead to greater reliability,

48

flexibility, and cost savings.

49

Many multiplex PCR methods have been reported previously, including

50

universal primer-multiplex PCR (UP-M-PCR) for the simultaneous detection of

51

pathogenic bacteria [3], common primer multiplex (CP-M-PCR) for the identification

52

of meat species [4], multiplex PCR for the identification of genetically modified (GM)

53

soybean events [5], microdroplet PCR implemented capillary gel electrophoresis

54

(MPIC) for the detection of multiple DNA targets [6], UP-M-PCR and capillary

55

electrophoresis–laser-induced fluorescence analysis for GM maize detection [7],

56

UP-M-PCR with sequencing gel electrophoresis for GM crops detection [8],

57

microarray multiplex detection [9] and multiplex ligation-dependent probe

58

amplification (MLPA) [10]. MPIC can simultaneously detect up to twenty-four targets,

59

thus providing the largest number of targets yet reported for multiplex PCR. However,

60

the twenty-four targets were pre-amplified separately into three groups of eight, which 3

61

could be regarded as eight-plex PCR.

62

Multiplex PCR obviously has many advantages compared to conditional PCR. It

63

is convenient, saves time and labor and is efficient for large-scale scientific, clinical,

64

and commercial applications, such as the detection of infectious microorganisms [11],

65

genetically modified organisms (GMO), or the diagnosis of infectious diseases [12].

66

In recent years, multiplex PCR has emerged as a core technology for high-throughput

67

SNP genotyping [13].

68

Although multiplex PCR has many advantages, it also has several disadvantages

69

that cannot be ignored, including self-inhibition among different sets of primers,

70

non-specific amplification and preferential amplification of partial target molecules

71

[14]. MLPA was devised to resolve these problems associated with conventional

72

multiplex PCR and has since widely used in medical diagnostics [15, 16]. Recently,

73

this method has also been used to detect GMOs [17-19]. Capillary electrophoresis is

74

always used as the detection apparatus for MLPA and other multiplex PCR methods.

75

MLPA with agarose gel detection method, which could simultaneously amplify many

76

exons of the DMD gene, has been reported. However, the use of agarose gel was

77

useful only when the amplicons to separate differ of at least 20-30 base pairs [20].

78

Moreover, the ligation probes are always labeled with fluorescent dyes. Therefore, the

79

detection cost is expensive and with high requirements of PCR products. Furthermore,

80

two universal PCR primers are needed to accomplish the amplification after the

81

ligation reaction, thus making the reaction system more complex. Meanwhile, the

82

detection device apparatus is often not compatible for the high-throughput detection 4

83

of complex samples.

84

In view of these shortcomings, we targeted seven of the most frequently used

85

transgenic marker genes, generic components, endogenous reference genes and

86

event-specific GMO genes to improve upon the existing multiplex PCR methods. Our

87

approach uses only a single universal primer for PCR amplification. We also took

88

advantage of PAGE to separate and analyze the PCR amplicons with single base pair

89

resolution [21]. Probes with different stuffer sequences were designed to test whether

90

the stuffer segments with different lengths affected the ligation rate or amplification

91

efficiency. This allowed us to develop an SUP-MLPA technique that is more accurate,

92

convenient, and scalable, thus providing a new method for high throughput detection

93

of multiple targets.

94

MATERIALS AND METHODS

95

Materials

96

Genetically modified maize MON863 and MON810 and genetically modified

97

soybean Roundup Ready soya (RRS) were supplied by Monsanto Company.

98

DNA extraction and purification

99

The grains were ground into powder with a blender. The powders were stored at

100

4°C before use. A total of 40~120 mg of each sample was measured for use with the

101

CTAB method [22]. The samples were then incubated with 2~3 µL of RNase at 37°C

102

for 20 min. The extracted DNA was purified using a Wizard DNA Purification Kit

103

(Promega, USA).

104

DNA concentrations were determined spectrophotometrically at 260 nm using a 5

105

UV/VIS spectrometer (Kontron, Neufahrn, Germany). DNA purity was determined by

106

calculating the 260/280 ratio

107

Ligation Probe and single universal primer sequences

108

The generic component gene CaMV 35S-promotor (35S) and nopaline synthase

109

(NOS), the maker gene neomycin phosphotransferase II (npt II), the endogenous

110

reference gene maize high mobility group protein (HMGa), the MON810

111

event-specific gene (MON810), the soya lectin (Lec) gene, and the Roundup Ready

112

soya inserted elements (RRS) were amplified in our study. The LPs and SUP (listed in

113

Table 1) were designed using ABI PRISM Primer Express Version 2.0 software

114

(Applied Biosystems Company, Foster City, CA, USA). These oligonucleotides were

115

synthesized by TAKARA (Dalian, China).

116

Hybridization of the ligation probes

117

The hybridization reactions were performed in 0.5 mL PCR reaction vessels

118

using a thermocycler (Applied Biosystems, USA). The reaction mixes (5µl total

119

volume) contained 10×Ampligase reaction buffer with 2 units Ampligase

120

(EPICENTRE, Madison, USA), 1µL of LP mix (10 nM) and 1.5µL template DNA

121

(100 ng/µL).

122

The initial DNA was denatured at 95°C for 3 min, followed by 20 denaturation

123

and ligation cycles at 95°C for 15 s and 63°C for 5 min. After ligation, the reaction

124

mixes were heated for 5 min at 98°C to inactivate the enzyme and were stored at 4°C.

125

PCR amplification

126

The reaction mix used for ligation product amplification (25 µl total volume) 6

127

contained 10× Ex Taq buffer with 1 unit Ex Taq polymerase, 12.5 µmol of each dNTP

128

(TAKARA, Dalian, China), 1µl universal primer (10 nM) and 1 µl template from the

129

ligation mix. DNA denaturation and polymerase activation at 95°C for 8 min were

130

followed by 45 amplification cycles at 95°C for 15 s and 58°C for 60 s, with a final

131

step of 7 min at 72°C. The reaction mixes were then cooled to 4°C.

132

Preparation of the polyacrylamide gel

133

An 8% polyacrylamide gel was prepared from 31.5 g urea, 15 mL 10×TBE, 40

134

mL 30% acrylamide stock solution and 150 mL distilled water. After filtering and

135

degassing the polyacrylamide solution, 150 µL TEMED and 150 µL 25% AP were

136

added [8]. The gel was cast in a vertical electrophoresis apparatus (Sequi-Gen® GT

137

Nucleic Acid Electrophoresis Cell, BIO-RAD, cat no.165-3862) according to the

138

manufacturer’s instructions.

139

Polyacrylamide gel electrophoresis

140

The power source was turned on half an hour before adding the sample to the gel

141

to remove bubbles and impurities from the gel. The gel was then loaded with 4 µL

142

samples and then immediately electrophoresed under a constant power of 60 W to

143

reduce sample diffusion and enhance band resolution. Gel electrophoresis was

144

continued until the desired fragment size separation was achieved. After

145

electrophoresis, the gels were silver stained [23].

146

Effect of different stuffer sequences

147

A series of LPs with different lengths of stuffer sequence were designed, the

148

stuffer sequences contained no homology with the corn and soybean genomes 7

149

according to a BLAST in NCBI GenBank. The stuffer sequence gradually increased

150

in size from Nos-F to Nos-6F (Table 2).

151

To assess the amplification efficiency of the different primers, quantitative PCR

152

(qPCR) was performed. The qPCR reaction was performed in a final volume of 25 µL,

153

containing 2.5×SYBR GREEN MIX (TIANGEN, CHINA), 2 µmol universal primer

154

and 1.5 µL template from the ligation reaction. The thermal cycling programs were

155

the same as described above.

156

RESULTS

157

Design of the ligation probes and the single universal primer

158

The annealing temperatures of the target-specific sequence on the ligation probes

159

(LPs) and the single universal primer (SUP) strongly influenced the efficiency and

160

specificity of our method, especially during the first ligation reaction. Both the space

161

structures of LPs and their interactions would affect the ligation efficiency; therefore,

162

the design parameters of the LPs were very strict. In the SUP-MLPA system, we used

163

only a single universal primer to conduct the PCR amplification; therefore, the

164

common sequences contained in the 5’end of the forward LP and the 3’ end of the

165

reverse LP were of reverse complementarity so that the universal primer could bind to

166

both. To prevent hybridization between the LPs themselves, the annealing

167

temperature of the target-specific sequences was higher than that of the common

168

sequences. Hence, the target-specific sequences hybridized with the templates first

169

during thermal cycling. A schematic of the LP design was shown in Figure 1.

170

All of the target-specific sequence on LPs had a Tm of 63°C, and the Tm of the 8

171

SUP was 58°C. The temperature difference of 5°C was enough for the ligation

172

reaction to happen. LPs and SUP with a 2°C difference in Tm were also designed, this

173

difference caused the amplification products to be barely visible on the PAGE gel,

174

and the LPs dimers were bright.

175

Feasibility of SUP-MLPA

176

For testing the feasibility of MLPA, single to hepta-plex ligation-dependent probe

177

amplifications were carried out to assess probe interference (Figure 2). Every band

178

could be clearly observed through the 8% polyacrylamide gel electrophoresis,

179

indicating that each target fragment was amplified effectively. The minimum

180

detectable difference between the adjacent targets was 3bp.

181

We also run the products on 5% and 10% polyacrylamide gel. In the 5%

182

polyacrylamide gel, the products were difficult to separate for the same

183

electrophoresis time as the 8% gels and the products were focused at the bottoms of

184

the gel. In 10% polyacrylamide gel, the products took too long time to separate

185

because of the high degree of gel cross-linking.

186

LP concentration

187

The sensitivity of MLPA depended on both the template concentration and the

188

ligation probe concentration. For our SUP-MLPA method, the sequences of LPs were

189

longer than those of traditional probes, and they contained the reversely

190

complementary sequences. Therefore, to prevent ligations and mismatches between

191

the LPs themselves, the concentrations of LPs required optimization. We chose three

192

probes (MON810, Lec and nptII) for separate hybridization with the templates for 9

193

MON810, RRS and MON863, respectively. A set of single ligation-dependent probes

194

amplifications were diluted 10-fold from 10 µM to 0.01 µM, resulting in an

195

amplification of the target product that was proportionate to the concentration (Figure

196

3).

197

The amounts of the amplified products decreased according to the concentrations

198

of the ligation probes. At 0.01 µM, no amplicons were observed for MON810 and Lec,

199

and very few amplicons were observed for nptII. Therefore, the lowest concentration

200

of ligation probe that was capable of detecting the target gene was 0.1 µM.

201

Addition of the PCR enhancer

202

The SUP-MLPA method used only one universal primer to amplify the template.

203

We wanted to further improve this system by adding DMSO and nano-gold particles

204

[24-26] to enhance or stabilize the reaction.

205

The enhancers were added separately in the ligation reactionstage and the PCR

206

amplification stage. The amplification conditions were performed using the same

207

thermal cycling parameters as before. However, after polyacrylamide gel

208

electrophoresis, the backgrounds were not clear and non-specific products appeared

209

(Supplementary Figure 1). We suspect that these enhancers are not compatible with

210

the Ampligase enzyme that we used in our system.

211

Sensitivity of the novel MLPA system

212

The sensitivity of SUP-MLPA method was assessed for a seven-plex PCR using

213

the mixture of samples. We performed separate serial dilutions using the mixture

214

samples (25 ng, 5 ng, 0.1 ng, 0.05 ng and 0.01ng), which resulting in a significantly 10

215

disproportionate amplification of target DNA. The amounts of the amplified products

216

decreased as the amounts of the templates decreased. For a target template amount of

217

0.05 ng (lanes 4, 9 and 14), only MON810 has little products, and for others no

218

amplicons were detected. Therefore, the detection limit for the target gene, as shown

219

in Figure 4, was 0.1 ng of target DNA per reaction. Compared with the published

220

conventional multiplex PCR, which detecting GM maize lines simultaneously [27],

221

the limit of detection was 0.25% GM in the total 100 ng template, equaling to 0.25 ng

222

GM content in the mixed samples, the novel SUP-MLPA has a higher sensitivity than

223

it.

224

Effects of different stuffer sequence lengths on the ligation probes

225

The amplification efficiency for different stuffer sequences was detected by

226

SYBR Green quantitative PCR (qPCR). After the first ligation reaction, each product

227

was amplified by SUP; therefore, the products can be regarded as the UP-M-PCR. In

228

the UP-M-PCR system, if the concentrations of the templates are the same, the PCR

229

amplification efficiencies of the different samples will also be the same. Therefore, if

230

we use qPCR as the detection method, the Ct value and amplification curve for each

231

sample will theoretically be the same, assuming with the equal template

232

concentrations.

233

All of the reaction reagents for qPCR were the same, with the exception of the

234

templates. Therefore, the different amplification efficiencies and amplification curves

235

must have been caused by different concentrations of the ligation products. The

236

differences in ligation product concentration were caused by the different ligation 11

237

efficiencies of the probes.

238

All of the Ct values were approximately 25, indicating that there were no

239

significant differences among the samples (Table 3). The melting temperatures

240

increased as the length of the stuffer sequence increased. Different ligation probes did

241

not lead to differences in the product concentrations, as observed for the first round of

242

ligation. This result indicated that we could change the length of the stuffer sequence

243

to distinguish many more targets that have similar lengths of target-specific

244

sequences.

245

DISCUSSION

246

Our study shows that the SUP-MLPA approach in combination with

247

polyacrylamide gel sequencing electrophoresis is applicable for detection of multiplex

248

targets. The design of LPs and SUP were very important for the SUP-MLPA

249

technique because the primer specificity and the melting temperature were more

250

critical than for conventional multiplex PCR.

251

This method usually uses a pair of universal primers to conduct the PCR

252

amplification. The probes are also always labeled with a fluorescent dye (FAM) and

253

with the capillary electrophoresis as the detection apparatus. The conventional

254

strategy is expensive and requires high quality of PCR products. In our study, we

255

developed a new method that avoids some of these drawbacks.

256

The UP-M-PCR method and sequencing gel electrophoresis method were

257

originally developed by our lab. In the present study, we used similar principles and

258

equipment to simultaneously detect seven target genes in GMOs. Polyacrylamide gels 12

259

allow the testing of relatively small DNA molecules (less than 1000 bp in length). The

260

products examined in our study were all approximately 100 bp. The lanes and the area

261

of the gel used for sequencing electrophoresis were both larger than for ordinary gels.

262

Through optimization, we found that the sequencing electrophoresis was most

263

appropriate for target detection.

264

Due to the range of the sequences which fit for designing target-specific

265

sequences was short, and the length of the target-specific sequences for all probes

266

maybe the same, it was not possible to rely on product size for the separation of the

267

amplicons. Hence, we studied the effect of different length of stuffer sequences on the

268

probes for the first time. Through our research, the length of the stuffer sequence did

269

not affect the ligation efficiency. Therefore, we were able to adjust the length of the

270

stuffer sequence to achieve separation based on the size of the products, thus

271

increasing the multiplicity of the assay. Any sequence meeting the requirements for

272

designing the LPs can be applied in this PCR system.

273

With the dramatic increase in the number of targets that can be simultaneously

274

amplified by PCR, it becomes urgent to improve the development of stable and

275

efficient multiplex methods, especially for applications involving GM crops. The

276

development of our simultaneous SUP-MLPA and sequencing gel electrophoresis

277

method allows the detection of seven commonly used selectable marker genes,

278

reporter genes and endogenous genes in one reaction. This detection method reduces

279

costs, is easily performed, and provides direct results. Moreover, this new

280

SUP-MLPA can be used in all the fields where relate to multiplex PCR, and has great 13

281

potential and application value.

282

ACKNOWLEDGEMENTS The study was funded by the National GMO Cultivation Major Project of New

283 284

Varieties (No. 2008ZX08012-001).

285

REFERENCES

286

1. Y. Zhang, D. Zhang, W. Li, J. Chen, Y. Peng, W. Cao, A novel real-time

287

quantitative PCR method using attached universal template probe, Nucleic Acids

288

Res. 31 (2003) e123.

289

2. M.E. Elfath, M.A. Ahmed, J.C. Robert, E.K. Paul, Multiplex PCR: optimization

290

and application in diagnostic virology, Clin. Microbiol. Rev. 134 (2000)

291

559–570.

292

3. Y. Yuan, W. Xu, Z. Zhai, H. Shi, Y. Luo, Z. Chen, K. Huang, Universal

293

primer-multiplex PCR approach for simultaneous detection of Escherichia coli,

294

Listeria monocytogenes, and Salmonella spp. in food samples, J. Sci. Food Agr.

295

74 (2009) M446-452.

296

4. W. Bai, W. Xu, K. Huang, Y. Yuan, S. Cao, Y. Luo, A novel common primer

297

multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat

298

species, Food Control 20 (2009) 366–370.

299

5. J.H. Kim, D. Jeong, Y.R. Kim, Y.K. Kwon, G.S. Rhee, D. Zhang, H.Y. Kim,

300

Development of a Multiplex PCR Method for Testing Six GM Soybean Events,

301

Food Control 31 (2012) 366–371.

302

6.

J. Guo, L. Yang, L. Chen,

D. Morisset, X. Li, L. Pan, D. Zhang, MPIC: a 14

303

high-throughput analytical method for multiple DNA targets, Anal. Chem. 83

304

(2011) 1579-1586.

305

7. C. Zhang, W. Xu, Z. Zhai, Y. Luo, X. Yan, N. Zhang, K. Huang, Universal

306

primer-multiplex-polymerase

chain

reaction

(UP-M-PCR)

307

electrophoresis–laser-induced

fluorescence

308

detection of six genetically modified maize lines, J. Sci. Food Agr. 59 (2011)

309

5188-5194.

analysis

for

the

and

capillary

simultaneous

310

8. W. Xu, Z. Zhai, K. Huang, N. Zhang, Y. Yuan, Y. Shang, Y. Luo, A novel

311

universal primer-multiplex-PCR method with sequencing gel electrophoresis

312

analysis, PLoS One 7 (2012) e22900.

313

9. S. Bierbaum, N. Königsfeld, N. Besazza, K. Blessing, G. Rücker, U. Kontny, R.

314

Berner, M. Schumacher, J. Forster, V. Falcone, C. van de Sand, A. Essig, D.

315

Huzly, G. Rohde, D. Neumann-Haefelin, M. Panning, Performance of a novel

316

microarray multiplex PCR for the detection of 23 respiratory pathogens

317

(SYMP-ARI study), Eur. J. Clin. Microbiol. Infect. Dis. 31 (2012) 2851-61.

318

10. J.P. Schouten, C.J. McElgunn, R. Waaijer, D. Zwijnenburg, F. Diepvens, G. Pals,

319

Relative

quantification

of

40

nucleic

acid

sequences

by

multiplex

320

ligation-dependent probe amplification, Nucleic Acids Res. 30 (2002) e57.

321

11. A. Pinar, N. Bozdemir, T. Kocagoz, R. Alacam, Rapid detection of bacterial

322

atypical pneumonia agents by multiplex PCR, Cent. Eur. J. Public Health 12

323

(2004) 3–5.

324

12. E. Elnifro, A. Ashshi, R. Cooper, P. Klapper, Multiplex PCR: optimization and 15

325

application in diagnostic virology, Clin. Microbiol. Rev. 13 (2000) 559–570.

326

13. S. Inagaki, Y. Yamamoto, Y. Doi, T. Takata, T. Ishikawa, K. Imabayashi, K.

327

Yoshitome, S. Miyaishi, H. Ishizu, A new 39-plex analysis method for SNPs

328

including 15 blood group loci, Forensic Sci. Int. 144 (2004) 45–57.

329

14. M. Chaouachi, G. Chupeau, A. Berard, H. McKhann, M. Romaniuk, S. Giancola,

330

V. Laval, Y. Bertheau, D. Brunel, A high-throughput multiplex method adapted

331

for GMO detection, J. Agric. Food Chem. 56 (2008) 11596–11606.

332

15. L. Véronèse, O. Tournilhac, P. Combes, N. Prie, E. Pierre-Eymard, R. Guièze, R.

333

Veyrat-Masson, J.O. Bay, P. Vago, A. Tchirkov, Contribution of MLPA to routine

334

diagnostic testing of recurrent genomic aberrations in chronic lymphocytic

335

leukemia, Cancer Genet. 206 (2013) 19-25.

336

16. J. Cui, M. Azimi, A.D. Adekile, H. Al Awadhi, C.C. Hoppe, Detection of

337

anti-Lepore Hb P-Nilotic by multiplex ligation-dependent probe amplification,

338

Hemoglobin 36 (2012) 276-282.

339

17. F. Moreano, A. Ehlert, U. Busch, K.H. Engel, Ligation-dependent probe

340

amplification for the simultaneous event-specific detection and relative

341

quantification of DNA from two genetically modified organisms, Eur. Food Res.

342

Technol. 222 (2006) 5-6.

343

18. A.L. Holck, S.M. Drømtorp, E. Heir, Quantitative, multiplex ligation-dependent

344

probe amplification for the determination of eight genetically modified maize

345

events, Eur. Food Res. Technol. 230 (2009) 185-194.

16

346

19. A. Ehlert, F. Moreano, U. Busch, K.H. Engel, Development of a modular system

347

for

detection

of

genetically

modified

organisms

in

food

based

on

348

ligation-dependent probe amplification. Eur. Food Res. Technol. 227 (2008)

349

805-812.

350

20. T. Lalic, R.H. Vossen, J. Coffa, J.P. Schouten, M. Guc-Scekic, D. Radivojevic, M.

351

Djurisic, M.H. Breuning, S.J. White, J.T. den Dunnen, Deletion and duplication

352

screening in the DMD gene using MLPA,Eur. J. Hum. Genet. 13 (2005)

353

1231-1234.

354 355 356 357 358 359 360 361 362 363

21. J. Sambrook, D.W. Russell, Molecular cloning: A laboratory manual (3rd ed.), Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001 22. C.M. Tsai, C.E. Frasch, A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels, Anal. Biochem. 119 (1982) 115-119. 23. R. J. Reece, Analysis of Genes and Genomes, John Wiley & Sons Inc., New York, 2004, pp. 89. 24. P.N. Hengen, Optimizing multiplex and LA-PCR with betaine, Trends Biochem. Sci. 22 (1997) 225-226. 25. T. Hung, K. Mak, K. Fong, A specificity enhancer for polymerase chain reaction, Nucleic Acids Res. 18 (1990) 4953.

364

26. H. Li, J. Huang, J. Lv, H. An, X. Zhang, Z. Zhang, C. Fan, J. Hu, Nanoparticle

365

PCR: Nanogold-Assisted PCR with Enhanced Specificity, Angew. Chem. Int.

366

Edit. 44 (2005) 5100-5103.

17

367

27. H.K. Shrestha, K. Hwu, S. Wang, L. Liu, M. Chang, Simultaneous detection of

368

eight genetically modified maize lines using a combination of event-and

369

construct-specific multiplex-PCR technique, J. Agric. Food Chem. 56 (2008)

370

8962–8968.

18

371

Figure Legends

372

Figure 1 Diagram of the LP design. The numbers 5’ and 3’ represent the direction of the

373

nucleotides. The single strand which 5’ and 3’ were in black represents the template after

374

denaturation. The strand in red represents the forward probe, and the strand in blue presents the

375

reverse probe. A, The ligation reaction can be conducted when the annealing temperature of the

376

target-specific sequence is higher than the common sequence, such that the probes hybridize with

377

the target. B, The probes can hybridized with themselves when the annealing temperature of the

378

target-specific sequence is lower than the common sequence.

379

Figure 2 Probe interference for single to hepta-plex SUP-MLPA.

380

the seven targets. Lanes 1’-7’, SUP-MLPA products starting from the largest amplicon (MON810,

381

110 bp) to the shortest amplicon (35S, 83 bp). B, The mix contains the templates with equivalent

382

DNAs from the three different crops. Lanes 1-7, PCR products starting from the largest amplicon

383

followed by the addition of a second primer pair until the seventh probe pair. The products in

384

order of size from largest to smallest were: MON810, 110 bp; RRS, 107 bp; HMGa, 102 bp; Lec,

385

96 bp; Nos, 91 bp; nptII, 86 bp; 35S, 83 bp. Lanes 8-13, PCR products starting with the seven-plex

386

amplicon, followed by the elimination of the largest amplicon primer pair, until the smallest

387

amplicon. M, the 50bp DNA Ladder.

388

Figure 3 Optimization of the ligation probe concentration for SUP-MLPA. The concentration

389

of the probes were 10-fold diluted from 10 µM to 0.01 µM. Lanes 1-4, the amplification results

390

for MON810 when the concentration of the probe was decreased in 10-fold increments from 10

391

µM to 0.01 µM. Lanes 5-8, the amplification results for Lec. Lanes 9-12, the amplification results

392

for npt II. 19

A, Single SUP-MLPA of

393

Figure 4 The detection limit of SUP-MLPA. The sensitivity of SUP-MLPA was assayed in

394

seven-plex using only one DNA template. The template is identified at the bottom of the figure.

395

The concentration of the template was serially diluted from 25 ng to 0.01 ng. Lanes 1-5, the

396

template for the MON810 crop for concentrations from 25 ng, 5 ng, 0.1 ng, 0.05 ng and 0.01ng.

397

Lanes 6-10, the template for the MON863 crop with the same serial dilution as MON810. Lanes

398

11-15, the template for RRS with the same serial dilution as MON810.

20

399

Figure 1 Diagram of the LP design

400

21

401

Figure 2 Probe interference for single to hepta-plex SUP-MLPA

402

22

403

Figure 3 Optimization of the ligation probe concentration for SUP-MLPA

404

23

405

Figure 4 The detection limit of SUP-MLPA

406

24

Table 1 The sequences of the LPs and SUP Target gene

Sequence

Product size (bp)

MON810-F

TTGGTCGTGGTGGTGGTTTcgtgtcacgttgacgatgtaccaatgcttcgaaggacgaaggactct 110

MON810-R

RRS-F

P*-aacgtttaacatcctttgccattgcAAACCACCACCACGACCAA

TTGGTCGTGGTGGTGGTTTcgtgtcacgttgcgatgtacaaacatagggaacccaaatggaaaag 107

RRS-R

P-gaaggtggctcctacaaatgccaAAACCACCACCACGACCAA

HMGa-F

TTGGTCGTGGTGGTGGTTTcagtgtcatgatgtacactgcagctaagaaggctcctgc

HMGa-R

P- caaggaggaagaggaggaagatgaaAAACCACCACCACGACCAA

102

Lec-F

TTGGTCGTGGTGGTGGTTTgacgatgtacccacgggactcgacatacctggg 96

Lec-R

P- gaatcgcatgacgtgctttcttgAAACCACCACCACGACCAA

Nos-F

TTGGTCGTGGTGGTGGTTTtcgttcaaacatttggcaataaagttt 91

Nos-R

P-cttaagattgaatcctgttgccggAAACCACCACCACGACCAA

nptⅡ-F

TTGGTCGTGGTGGTGGTTTcatagcgttggctacccgtgatatt 86

nptⅡ-R

P-gctgaagagcttggtggcgaaAAACCACCACCACGACCAA

35S-F

TTGGTCGTGGTGGTGGTTtgggatgacgcacaatcccacta

35S-R

P-tccttcgcaagacccttcctcAAACCACCACCACGACCAA

83

UP

TTGGTCGTGGTGGTGGTTT

* P- present for the phosphorylation

25

Table 2 The sequences of the LPs with different length of stuffer sequences for NOS Target gene

Sequence

Product size(bp)

NOS- F

TTGGTCGTGGTGGTGGTTTtcgttcaaacatttggcaataaagttt

91

NOS-1F

TTGGTCGTGGTGGTGGTTTatgtcgttcaaacatttggcaataaagttt

94

NOS-2F

TTGGTCGTGGTGGTGGTTTatgatgtcgttcaaacatttggcaataaagttt

97

NOS-3F

TTGGTCGTGGTGGTGGTTTatgatgatgtcgttcaaacatttggcaataaagttt

100

NOS-4F

TTGGTCGTGGTGGTGGTTTatgatgatgatgtcgttcaaacatttggcaataaagttt

103

NOS-5F

TTGGTCGTGGTGGTGGTTTatgatgatgatgatgatgtcgttcaaacatttggcaataaagttt

109

NOS-6F

TTGGTCGTGGTGGTGGTTTatgatgatgatgatgatgatgatgtcgttcaaacatttggcaataaagttt

115

NOS-R

P-cttaagattgaatcctgttgccggAAACCACCACCACGACCAA

* P- present for the phosphorylation

26

Table 3 The qPCR results for the LPs with different stuffer sequences Ct Value Ligation probe

Ct Mean

Tm

1

2

Nos

25.025

25.067

25.046

81.603

nos-1

25.147

25.269

25.208

81.618

nos-2

25.439

25.618

25.529

81.686

nos-3

25.454

25.561

25.508

81.702

nos-4

25.597

25.423

25.510

81.752

nos-5

25.175

25.402

25.289

81.816

nos-6

25.907

25.494

25.701

81.922

nos-7

26.018

25.833

25.926

81.998

27