臺灣博碩士論文加值系統

針對生物去氧核醣核酸 (DNA)進行序列的分析時，會先採集實驗對象的DNA，在大多數的情況下，DNA樣本常常不足以用來做大量重複性的實驗，所以必須透過DNA擴增技術將目標基因或序列進行擴增，以利後續實驗分析所需。目前大部分的DNA擴增技術例如聚合酶連鎖反應等，都需仰賴熱循環機器將DNA進行變性 (Denaturation)、 接合 (Annealing)、延伸 (Extension)等過程，並且需要精準控制每個過程的溫度上升、下降，如此循環，才能得到擴增後大量的產物。恆溫重組聚合酶去氧核醣核酸擴增等技術是在2006年TwistDx公司所研發，該技術是使引子序列與酵素形成複合體，該複合體會在DNA模板上尋找同源序列的位置，將DNA模板解旋，接著使用重組聚合酶來進行擴增，整個過程的溫度大約維持在37 ~ 42°C，這將使得DNA擴增技術得到一個新突破，恆溫重組聚合酶去氧核醣核酸擴增技術將不再需仰賴熱循環機器，提升DNA擴增技術的攜帶性、方便性。然而，該技術最關鍵的技術在於引子要如何設計才能使得恆溫重組聚合酶去氧核醣核酸擴增正確擴增該目標基因或序列，在進行多重引子設計時，還需要避免兩兩引子因為序列過度相似而導致形成引子二聚體從而降低擴增效率。本研究針對使用該家公司所研發實驗套組的文獻，將文獻中所作者設計與使用的恆溫重組聚合酶去氧核醣核酸擴增引子組，進行蒐集、統計出引子特徵值的分佈、整合文獻中提到設計引子的建議，接著使用一系列生物資訊方法，例如使用Primer3依照恆溫重組聚合酶去氧核醣核酸擴增的特徵產生候選引子對，再藉由Bowtie 進行序列比對，確認每一組引子對的專一性，搭配遺傳演算法實現最佳化找出兩兩引子之間溫度不會過高而形成二聚體的組合，設計出多重恆溫重組聚合酶去氧核醣核酸擴增引子組。最後，本研究以此平台分別設計出多重恆溫重組聚合酶核酸擴增與多重聚合酶連鎖反應的引子對提供未來實驗驗證如凝膠電泳、次世代定序或是Nanopore MinION定序平台。總而言之，本研究將此技術建立成網頁平台與單機版的程式，提供未來使用者能夠輸入參數並且自動的搭配本研究整合的恆溫重組聚合酶去氧核醣核酸擴增引子特徵，設計出符合自己實驗需求的多重聚合酶連鎖反應或是多重恆溫重組聚合酶去氧核醣核酸擴增的引子組。

At present, most of the deoxyribonucleic acid (DNA) amplification techniques such as polymerase chain reaction (PCR). PCR relies on the thermal cycle machine, through denaturation, annealing, extension, the process requires precise control the temperature. Recombinase polymerase amplification (RPA) technology is developed by TwistDx in 2006. First, it makes the primer sequence and protein form a complex, the complex will find the location of the homologous sequence on the DNA template and open double-stranded DNA helix structure. Next, amplification was performed by recombinase polymerase. The temperature of the whole process is maintained at about 37 to 42°C, which will allow the DNA amplification technology get a new breakthrough. DNA amplification technology at constant temperature will no longer need to rely on the thermal cycle machine, enhances this DNA amplification technology’s portability and convenience. However, the most important part of the technology is how to design primers to make the RPA correctly amplify the target gene or sequence. In addition, design primers for multiplex PCR or RPA, it needs to avoid the two primers because the sequence with excessive similarity leads to form primer dimers so that reduce the amplification efficiency. So far, it is still not found that someone provides a primer design for multiplex RPA platform. In this study, we collect RPA primers from literature, and statistics out RPA primer features and integrate the recommendations of primer design from literature. Next, according to as above, use a series of bioinformatics methods like we use Primer3 to generate candidate primer groups, and then we use Bowtie to confirm the specificity of each primer pairs. Finally, the genetic algorithm was used to find out optimized primer group that the temperature between the two primers will not be too high to form primer dimers. In this study, we respectively designed primer sets for multiplex PCR and multiplex RPA to provide future experimental verification, such as gel electrophoresis, next-generation sequencing or Nanopore MinION sequencing platform. In summary, this study develops a web platform and a standalone tool allows users to design multiplex PCR or RPA primer sets that meet their own experimental needs.

摘要 i
ABSTRACT ii
誌謝 iii
Table of contents iv
List of figures vi
List of tables viii
Chapter 1 Introduction 1
1.1 Overview 1
1.2 Background 6
1.2.1 DNA-based diagnostics 6
1.2.2 Polymerase chain reaction (PCR) 7
1.2.3 Recombinase polymerase amplification 8
1.2.4 Genetic variation 10
1.2.5 Single nucleotide polymorphism 10
1.2.6 SNP Genotyping 11
1.3 Primer design 13
1.3.1 What are primers? 13
1.3.2 What are primer dimers? 15
1.3.3 Primer design for Multiplex Polymerase Chain Reaction 15
1.4 Sequencing technologies 19
1.4.1 Next generation sequencing 19
1.4.2 Nanopore MinION sequencing platform 20
1.5 Motivation 21
1.6 Specific aims 22
Chapter 2 Related works 23
Chapter 3 Materials and Methods 25
3.1 Workflow of this study 25
3.2 Collect RPA primers from literature 26
3.3 Feature distribution statistics 27
3.4 Considerations for Primer design 28
3.5 Primer design parameters for produce RPA primer candidates 33
3.6 Using Primer3-2.3.7 produce primer candidates 34
3.7 Check primer specificity using Bowtie-1.2.1.1 35
3.8 Produce optimal primer set by genetic algorithm Using ntthal 36
3.9 Develop a website allow user design primer 39
3.10 Develop a standalone tool allow user design primer 44
Chapter 4 Results 45
4.1 Developed website results 45
4.2 Developed standalone tool results 48
4.3 Primer design for Multiplex Polymerase Chain Reaction 50
4.4 Primer design for Multiplex Recombinase Polymerase Amplification 53
Chapter 5 Conclusion and Discussion 56
5.1 Summary, advantage, and application 56
5.2 Weaknesses, and limitations 57
5.3 Compared with others primer design platforms 59
5.4 Future works 59
Reference 61
Appendix 64
1. Collection of recombinase polymerase amplification primers list 64

1. Kunze A, Dilcher M, Abd El Wahed A, Hufert F, Niessner R, Seidel M: On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria. Anal Chem 2016, 88(1):898-905.
2. Wang J, Liu L, Li R, Wang J, Fu Q, Yuan W: Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplification. Arch Virol 2016, 161(4):1015-1018.
3. Wang R, Zhang F, Wang L, Qian W, Qian C, Wu J, Ying Y: Instant, Visual, and Instrument-Free Method for On-Site Screening of GTS 40-3-2 Soybean Based on Body-Heat Triggered Recombinase Polymerase Amplification. Anal Chem 2017, 89(8):4413-4418.
4. Londono MA, Harmon CL, Polston JE: Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics. Virol J 2016, 13:48.
5. Lau HY, Wang Y, Wee EJ, Botella JR, Trau M: Field Demonstration of a Multiplexed Point-of-Care Diagnostic Platform for Plant Pathogens. Anal Chem 2016, 88(16):8074-8081.
6. Yehia N, Arafa AS, Abd El Wahed A, El-Sanousi AA, Weidmann M, Shalaby MA: Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection. J Virol Methods 2015, 223:45-49.
7. Liu Y, Lei T, Liu Z, Kuang Y, Lyu J, Wang Q: A Novel Technique to Detect EGFR Mutations in Lung Cancer. Int J Mol Sci 2016, 17(5).
8. Kersting S, Rausch V, Bier FF, von Nickisch-Rosenegk M: Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens. Mikrochim Acta 2014, 181(13-14):1715-1723.
9. Dillon B, Thomas L, Mohmand G, Zelynski A, Iredell J: Multiplex PCR for screening of integrons in bacterial lysates. J Microbiol Methods 2005, 62(2):221-232.
10. Myers FB, Henrikson RH, Xu L, Lee LP: A point-of-care instrument for rapid multiplexed pathogen genotyping. Conf Proc IEEE Eng Med Biol Soc 2011, 2011:3668-3671.
11. DNA-based Molecular Diagnostic Techniques: Research Needs for Standardization and Validation of the Detection of Aquatic Animal Pathogens and Diseases.
12. Aebischer A, Wernike K, Hoffmann B, Beer M: Rapid genome detection of Schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR. J Clin Microbiol 2014, 52(6):1883-1892.
13. Teoh BT, Sam SS, Tan KK, Danlami MB, Shu MH, Johari J, Hooi PS, Brooks D, Piepenburg O, Nentwich O et al: Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification. J Clin Microbiol 2015, 53(3):830-837.
14. Sabate del Rio J, Steylaerts T, Henry OY, Bienstman P, Stakenborg T, Van Roy W, O'Sullivan CK: Real-time and label-free ring-resonator monitoring of solid-phase recombinase polymerase amplification. Biosens Bioelectron 2015, 73:130-137.
15. Yan L, Zhou J, Zheng Y, Gamson AS, Roembke BT, Nakayama S, Sintim HO: Isothermal amplified detection of DNA and RNA. Mol Biosyst 2014, 10(5):970-1003.
16. Zaghloul H, El-Shahat M: Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis. World J Hepatol 2014, 6(12):916-922.
17. Deng H, Gao Z: Bioanalytical applications of isothermal nucleic acid amplification techniques. Anal Chim Acta 2015, 853:30-45.
18. Piepenburg O, Williams CH, Stemple DL, Armes NA: DNA detection using recombination proteins. PLoS Biol 2006, 4(7):e204.
19. Boyle DS, McNerney R, Teng Low H, Leader BT, Perez-Osorio AC, Meyer JC, O'Sullivan DM, Brooks DG, Piepenburg O, Forrest MS: Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification. PLoS One 2014, 9(8):e103091.
20. Daher RK, Stewart G, Boissinot M, Bergeron MG: Recombinase Polymerase Amplification for Diagnostic Applications. Clin Chem 2016, 62(7):947-958.
21. Del Rio JS, Lobato IM, Mayboroda O, Katakis I, O'Sullivan CK: Enhanced solid-phase recombinase polymerase amplification and electrochemical detection. Anal Bioanal Chem 2017, 409(12):3261-3269.
22. Mekuria TA, Zhang S, Eastwell KC: Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification. J Virol Methods 2014, 205:24-30.
23. Yang Y, Qin X, Song Y, Zhang W, Hu G, Dou Y, Li Y, Zhang Z: Development of real-time and lateral flow strip reverse transcription recombinase polymerase Amplification assays for rapid detection of peste des petits ruminants virus. Virol J 2017, 14(1):24.
24. Crannell Z, Castellanos-Gonzalez A, Nair G, Mejia R, White AC, Richards-Kortum R: Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa. Anal Chem 2016, 88(3):1610-1616.
25. Chan K, Weaver SC, Wong PY, Lie S, Wang E, Guerbois M, Vayugundla SP, Wong S: Rapid, Affordable and Portable Medium-Throughput Molecular Device for Zika Virus. Sci Rep 2016, 6:38223.
26. PCR Primer Design Guidelines.
27. Rouchka EC, Khalyfa A, Cooper NG: MPrime: efficient large scale multiple primer and oligonucleotide design for customized gene microarrays. BMC Bioinformatics 2005, 6:175.
28. James A, Macdonald J: Recombinase polymerase amplification: Emergence as a critical molecular technology for rapid, low-resource diagnostics. Expert Rev Mol Diagn 2015, 15(11):1475-1489.
29. Rozen S, Skaletsky H: Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 2000, 132:365-386.
30. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG: Primer3--new capabilities and interfaces. Nucleic Acids Res 2012, 40(15):e115.
31. Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA: Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 2007, 35(Web Server issue):W71-74.
32. Langmead B, Trapnell C, Pop M, Salzberg SL: Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 2009, 10(3):R25.