Studies on the Nucleic Acid based Qualitative and Quantitative Methods of Analysis for Biotech Plants : 생명공학 식물의 핵산기반 정성 및 정량 분석방법에 관한 연구

다국어 초록 (Multilingual Abstract)

ABSTRACT

Biotech plants have continuously increased every year in the world. Although there is no commercial cultivation area for biotech plants in Korea, our country has absolutely depended on imports with the exception of rice for most of the crops such as maize, soybean, cotton and canola. Some consumers have worried about the potential risk, that is, the biotech plants may affect human health and ecological environment. Many countries have made their own labeling systems for biotech products. Korea also has its own GMO (genetically modified organism) labeling system. The purpose of GMO labeling system is to inform consumers of the presence of the biotech products in the plants or derived-products and therefore to help the consumers to choose their preferred products.
As a management tool, it is necessary to develop analytical methods to control GMO labeling system or LMO (living modified organism) management. The polymerase chain reaction (PCR) is the most efficient nucleic acid based analytical method to identify and quantitate biotech plants. There are four types of analytical approaches; screening, gene-specific, construct-specific and event-specific analysis. Screening and gene-specific methods are applicable to detect many events of biotech plants using common elements; promoter, terminator and a specific trait gene. These methods may be used to detect unapproved biotech plants or unknown biotech plants. The construct-specific method detects the junction region of two gene constructs as an intermediate between gene-specific and event-specific methods. In case of gene-specific and/or construct-specific methods, sometimes it is difficult to distinguish different events because some biotech plant events share the same genetic elements or vector DNA. The event-specific method has the highest specificity for one event by the junction region between the inserted DNA and the host genome.
In this study, the screening, gene-specific, construct-specific and event-specific analytical methods have been developed for eight events of biotech maize, four events of biotech cotton, four events of biotech canola and one event of biotech soybean. The screening method has been developed using common promoter and terminator of biotech plants for rapid detection in one reaction. The gene-specific method has been used for DNA chip. The construct-specific method has been derived from two other DNA constructs and the event-specific method has been derived from the junction region between the inserted DNA and the host genome.
The following methods have been studied. First, duplex screening method by CaMV35S promoter (P35S) and NOS terminator (tNOS) for biotech maize eight events NK 603, TC 1507, MON 863, MIR 604, Event 3272, MON 88017, LY 038 and DAS-59122-7. Second, gene-specific qualitative method using DNA chip for four kinds of biotech plants by four endogenous genes zSSⅡb, fsACP, Hmg, Le1; and nine introduced genes P35S, tNOS, pat, bar, epsps1, epsps2, pmi, cry1Ac, cry3B. Third, construct-specific qualitative and quantitative methods for biotech maize three events NK 603, TC 1507, MON 863; biotech cotton four events MON 531, MON 1445, MON 15985, MON 88913; and biotech canola four events GT 73, MS 8, RF 3, MS 8/RF 3. Fourth, event-specific qualitative and quantitative methods for biotech maize five events MIR 604, Event 3272, MON 88017, LY 038, DAS-59122-7; and biotech soybean one event MON 89788. Fifth, multiplex qualitative PCR method for biotech maize five events MIR 604, Event 3272, MON 88017, LY 038, DAS-59122-7; and duplex real-time PCR method for LY 038 in one reaction simultaneously.
The simplex qualitative analytical methods were confirmed the specificity by the single PCR product and the sensitivity was 0.01 % or 0.05 % as a relative limit of detection (LOD) and 0.005 ng or 0.025 ng as an absolute LOD, respectively. The multiplex qualitative analytical method was also confirmed the specificity and 0.5 % (0.25 ng) of relative (absolute) LOD for five biotech maize events. I also conftirmed the duplex screening PCR and DNA chip can be applicable for qualitative analysis.
The simplex and duplex quantitative methods were also developed using real-time PCR. As reference molecules, six standard plasmids were constructed from taxon-specific DNA sequences of four plants and construct-specific or event-specific DNA sequences of biotech plant events. In-house validation for simplex and duplex quantitative methods has been performed using six levels of mixing samples, 0.1 to 10.0 %. As results, the biases from the true value and the relative standard deviations (RSDs) were almost within the range of ±30 %. Limits of quantitation (LOQs) of the quantitative methods were 0.1 % or 0.5 % for simplex real-time PCRs for fifteen events of biotech plants and 0.5 % for duplex real-time PCR for LY 038.
Consequently, I report that these screening, gene-specific, construct-specific and event-specific analytical methods can be applicable for qualitative and quantitative analysis for biotech maize, cotton, canola and soybean.

Keywords: biotech plants; genetically modified organism (GMO); living modified organism (LMO); polymerase chain reaction (PCR); screening; gene-specific; construct-specific; event-specific; DNA chip; real-time PCR; simplex; duplex; multiplex; qualitative; quantitative; limit of detection (LOD); limit of quantitation (LOQ); reference molecule; standard plasmid

목차 (Table of Contents)

• Contents
• Abstract ⅰ
• List of Tables ⅶ
• List of Figures ⅸ
• Key to Abbreviatios xii
• Ⅰ. Introduction 1
• Ⅱ. Literature Review 5
• 1. Biotech Plants 5
• 2. Risk Assessment 6
• 3. Regulations 7
• 4. Analytical Methods 9
• Ⅲ. Materials and Methods 12
• 1. Materials 12
• 1.1 Maize 12
• 1.2. Cotton 12
• 1.3. Canola 12
• 1.4. Soybean 13
• 2. Methods 13
• 2.1. DNA Extraction 13
• 2.2. PCR Inhibitor Check 13
• 2.3. Junction Region Analysis 14
• 2.4. Oligonucleotide Primers and Probes 14
• 2.5. Taxon-Specific Reference Genes 14
• 2.6. Simplex PCR 15
• 2.6.1. Qualitative PCR Analysis 15
• 2.6.2. Quantitative PCR Analysis 16
• 2.6.3. Maize 16
• 2.6.3.1. Construct-Specific PCR 16
• 2.6.3.2. Event-Specific PCR 19
• 2.6.4. Cotton 22
• 2.6.4.1. Construct-Specific PCR 22
• 2.6.5. Canola 25
• 2.6.5.1. Construct-Specific PCR 25
• 2.6.6. Soybean 28
• 2.6.6.1. Event-Specific PCR 28
• 2.7. Multiplex PCR 29
• 2.7.1. Duplex Screening PCR 29
• 2.7.2. Multiplex Event-Specific PCR 29
• 2.7.3. Duplex Real-Time PCR 32
• 2.8. Standard Plasmid as a Reference Molecule 33
• 2.9. In-House Validation 35
• 2.10. DNA Chip 36
• 2.10.1. Gene-Specific PCR Primers and Probes 36
• 2.10.2. Microarray Construction 38
• 2.10.3. Labeling with Syto61 39
• 2.10.4. Multiplex PCR with Cy5-dCTP Labeling 39
• 2.10.5. Hybridization 40
• 2.10.6. Signal Detection 40
• Ⅳ. Results 41
• 1. Simplex PCR 41
• 1.1. Maize 41
• 1.1.1. Construct-Specific PCR 41
• 1.1.2. Event-Specific PCR 51
• 1.2. Cotton 65
• 1.2.1. Construct-Specific PCR 65
• 1.3. Canola 75
• 1.3.1. Construct-Specific PCR 75
• 1.4. Soybean 83
• 1.4.1. Event-Specific PCR 83
• 2. Multiplex PCR 90
• 2.1. Duplex Screening PCR 90
• 2.2. Multiplex Event-Specific PCR 91
• 2.3. Duplex Real-Time PCR 92
• 3. DNA Chip 99
• 3.1. Gene-Specific DNA Chip 99
• 3.2. Hybridization by Multiplex PCRⅠ 102
• 3.3. Hybridization by Multiplex PCRⅡ 106
• Ⅴ. Discussion 108
• Ⅵ. Literature Cited 113
• 국문요약 122
• 감사의 글 126