Comparative Evaluation of Three Extraction Methods for DNA

Comparative Evaluation of Three Extraction Methods for DNA Quantification and PCR Detection in Cocoa and Cocoa-Derived Products Lam Thi Viet Haa, Lore Vanlerberghea, Ha Thanh Toanb, Koen Dewettincka and Kathy Messensa a. Ghent University, b. Cantho University INTRODUCTION Several studies have reported the efficiency of using cocoa leaf material for the extraction of DNA. These different authors proved the efficiency of DNA extraction methods for cocoa studies and most of these methods were used for cocoa leaf tissues. Thus, to the best of our knowledge, alternative methods for extracting DNA from different cocoa-derived products have yet to be published. In this research, the DNA of cocoa-derived products was extracted with three different DNA extraction procedures, such as the commercial kits QIAamp. DNA Stool kit, Wizard Genomic DNA purification kit, and CTAB (cetyltrimethylammonium bromide) based protocols CTAB-Proteinase K. Three PCR primer pairs were selected to detect the presence of specific genes in cocoa derived products and to target a fragment of the chloroplast DNA, the 5 S r. DNA and the Seed Storage Protein (SSP) (Taberlet et al. , 1992; Petilliard & Crouzillat, 2004). The efficiency of these methods was evaluated by the purity, quantity, and amplification of the extracted DNA. METHODS CTABProteinase K procedure MATERIALS: Cocoa-derived products: cocoa leaves, cocoa beans, cocoa powder, cocoa butter, cocoa mass, and dark chocolate. - CTAB-Proteinase K (Gryson et al. , 2007) - QIAamp DNA Stool kit (Qiagen/Westburg, Leusden, the Netherlands), Wizard Genomic DNA kit QIAamp DNA Stool kit 1. OD measurement 2. PCR reaction - Wizard Genomic DNA purification kit (Promega, Madison, WI, USA) - The primer pair: plant c (5’-CGA AAT CGG TAG ACG CTA CG-3’) and plant d (5’-GGG GAT AGA GGG ACT TGA AC-3’) (Taberlet et al. , 1991). - The primers: 5 S I (5’-TTT AGT GCT GGT ATG ATC GC-3’) and 5 S II (5’-TGG GAA GTC CTC GTG TTG CA-3’); SSP III (5’-GGC AAT TTA CTT CGT GAC AAA CG-3’) and SSP IV (5’-CTC ATA TTT GCC AGG AGA ATT AC-3’) (Petiard and Crouzillat, 2004) RESULTS Table 1. Comparison of DNA yield, purity and PCR results for cocoa leaves and cocoa beans isolated with different methods Sample Extraction method DNA concentration A 260/280 range PCR amplification (ng/µL) Cocoa leaves Plant c/d 5 S I/II SSP III/IV QIAamp DNA Stool kit 25. 30 ± 1. 21 1. 4 -1. 7 + + - Wizard Genomic DNA kit 149. 98 ± 151. 72 1. 7 -2. 0 - - - CTAB-Proteinase K 116. 83 ± 5. 71 1. 7 -2. 0 + + + QIAamp DNA Stool kit 17. 99 ± 2. 81 1. 7 -2. 0 - - - Wizard Genomic DNA kit 292. 41 ± 36. 39 < 1. 4 - - - CTAB-Proteinase K 58. 37 ± 21. 78 >2. 0 + + - QIAamp DNA Stool kit 26. 25 ± 3. 45 1. 7 -2. 0 - - - Wizard Genomic DNA 831. 85 ± 220. 16 < 1. 4 - - - CTAB-Proteinase K 75. 32 ± 34. 37 1. 7 -2. 0 + + - Cocoa butter QIAamp DNA Stool kit 7. 00 ± 7. 07 1. 4 -1. 7 - - - Wizard Genomic DNA 6. 00 ± 5. 66 1. 4 -1. 7 - - - CTAB-Proteinase K 6. 00 ± 0. 00 ND - - - QIAamp DNA Stool kit 28. 12 ± 1. 68 1. 7 -2. 0 + + - Wizard Genomic DNA 507. 50 ± 5. 23 < 1. 4 - - - CTAB-Proteinase K 172. 1± 235. 47 1. 7 -2. 0 + - QIAamp DNA Stool kit 16. 61 ± 0. 16 1. 4 -1. 7 - - Wizard Genomic DNA 112. 57 ± 28. 75 < 1. 4 - - CTAB-Proteinase K 11. 83 ± 0. 24 < 1. 4 + - Cocoa beans Cocoa powder Cocoa mass Dark chocolate + Fig. 1: PCR results of DNA cocoa matrices extracted with CTAB-Proteinase K method using primer pairs plant c/d (a) and 5 S I/II (b): lanes 1 and 18, lambda DNA/Hind. III marker; lanes 2 and 3, cocoa leaves; lanes 4 and 5, cacao beans; lanes 6 and 7, cocoa powder; lanes 8 and 9, cocoa butter; lanes 10 and 11, cocoa mass; lanes 12 and 13, dark chocolate; lane 14, positive control DNA extraction (soya); lane 15, negative control DNA extraction (m. Q); lane 16, positive PCR control (soya DNA); lane 17, negative PCR control (MQ). +, positive amplification - , negative amplification +/-, detectable band ND, not detected Fig. 2: PCR results of DNA cocoa matrices extracted with Wizard Genomic DNAkit (a) and Wizard Genomic DNAkit after clean-up (b) methods using primers plant c and d: lanes 1, 18 and 35, DNA ladder; lanes 2, 3, 19 and 20, cocoa leaves; lanes 4, 5, 21 and 22, cacao beans; lanes 6, 7, 23 and 24, cocoa powder; lanes 8, 9, 25 and 26, cocoa butter; lanes 10, 11, 27 and 28, cocoa mass; lanes 12, 13, 29 and 30, dark chocolate; lanes 14 and 31, positive control DNA extraction (soya); lanes 15 and 32, negative control DNA extraction (m. Q); lanes 16 and 33, positive PCR control (soya DNA); lanes 17 and 34, negative PCR control (MQ). CONCLUSIONS 1. • • • The Wizard Genomic DNA kit and the CTAB-Proteinase K method were found to be the best for DNA quality and PCR detection in cocoa leaves. CTAB-Proteinase K procedure is highly recommended for cocoa beans Wizard Genomic DNA kit revealed the best PCR performance when applied to cocoa powder CTAB-Proteinase K method showed the good result for PCR detection of cocoa mass. The Wizard Genomic DNA kit showed the best PCR performance for dark chocolate The cocoa butter matrix gave the lowest DNA yield, purity, and amplification results for all the examined extraction methods Faculty of Bioscience Engineering – Laboratory of Food Technology and Engineering Contact person: Lam Thi Viet Ha (+84 945227097) Coupure links 653, 9000 Ghent, Belgium, tel. +32 9 264 61 62, fax +32 9 264 62 18 http: //www. fte. ugent. be vietha. lamthi@ugent. be