Downloaded 300 times
Uploaded byAref Farokhi Fard
Isothermal Nucleic Acid Amplification Techniques
This document provides an overview of various isothermal nucleic acid amplification techniques, including: 1. TMA, NASBA, SMART, SDA, RCA, LAMP, and others that amplify nucleic acids under isothermal conditions to overcome limitations of PCR like high costs and contamination risks. 2. It describes several techniques in detail, explaining their mechanisms which involve primers, polymerases, and isothermal reactions to exponentially amplify target sequences. 3. The techniques have applications in research, medicine, forensics and more due to their simplicity, lower costs, and ability to be used at point-of-care without specialized equipment.
More Related Content
Similar to Isothermal Nucleic Acid Amplification Techniques
![GENE_AMPLIFICATION_PROCESSES-ADV_BIOTECH_ FINAL SLIDES 2682025[1].pptx](https://cdn.slidesharecdn.com/ss_thumbnails/geneamplificationprocesses-advbiotechfinalslides26820251-250929051903-b02bcb19-thumbnail.jpg?width=640&height=640&fit=bounds)
Isothermal Nucleic Acid Amplification Techniques
- 1.
- 2. Isothermal Nucleic AcidAmplification Techniques 2
- 3.
- 4. TMA : Transcriptionmediated amplification NASBA : Nucleic acid sequence based amplification SMART : Signal mediated amplification of RNA technology SDA : Strand displacement amplification RCA : Rolling circle amplification MPRCA : Multiply-primed rolling circle amplification LAMP : Loop-mediated isothermal amplification IMDA : Isothermal multiple displacement amplification HDA : Helicase-dependent amplification SPIA : Single primer isothermal amplification cHDA : Circular Helicase-dependent amplification RAM : Ramification amplification method 4
- 5. 3WJ : Three-wayjunction S1-ext : S1 primer extension product SSB : Single-stranded DNA binding protein ELISA : Enzyme-linked immunosorbent assay ELOSA : Enzyme-linked oligosorbent assay ECL : Electrochemiluminescence POCT : Point-of-care testing or bed-side testing is defined as medical testing at or near the site of patient care Φ29 Pol : DNA Polymerase from the Bacillus subtilis phage phi29 with exceptional strand displacement and processive synthesis properties and inherent 3´→5' proofreading exonuclease activity SS : single strand NA : nucleic acid Pol: polymerase RE : restriction enzyme exo-def : exonuclease deficient Kbps : kilo base pairs 3SR : self-sustained sequence replication 5
- 6. NA amplificationis a pivotal process in biotech and molecular biology and has been widely used in research, medicine, agriculture and forensics. PCR is the preferred method but has limitations, including: 1. High cost of equipment 2. Contamination chances 3. Sensitivity to certain classes of contaminants and inhibitors 4. Can not be use in POCT 6
- 7. RNA polto make RNA from a promoter engineered in the primer RNase H removes the RNA from cDNA without the heat- denaturation, Thus the thermocycling step has been eliminated, generating an isothermal amplification method named 3SR FDA has approved the technique in NucliSence formulation (NASBAECL) for molecular detection of some microorganisms such as HCV and HIV-1 7
- 8. 1. Gel electrophoresis 2.Fluorescence probes (real-time NASBA) 3. Colorimetric assay (NASBA-ELISA) 4. ECL 8
- 9.
- 10.
- 11. The targetsequence is not itself amplified Based on the formation of 3WJ Two probes that hybridize to the target at adjacent positions The overlap between the two probes is only 8 bp The 3’ ends of the templates and facilitators are blocked to prevent extension Bst DNA pol extends the short probe to produce a ds T7 promoter 11
- 12.
- 13. T7 RNApol generates a signal, RNA1 (3) RNA1 anneals to a second template (RNA amplification probe) This leads to further extension and transcription by the DNA and RNA polymerases to generate increased amounts of a second signal, RNA2 (4) 13
- 14.
- 15. The enddetection method uses a 96-well format, measuring color change in a standard plate reader Therefore, multiple samples may be quantified simultaneously with no need for gel analysis 15
- 16. Phi 29DNA pol extends a circle-hybridized primer by continuously progressing around the circular DNA probe of several dozen nucleotides The SS nature of amplicons in case of linear RCA may be beneficial for subsequent manipulations 16
- 17. 17 Ligation reaction ofpadlock probes on RNA templates is not as efficient as on DNA templates Therefore it may be difficult to utilize conventional RCA for RNA detection yet Biosensors 2013, 3, 18-43
- 18. MPRCA usesphi29 pol and random primers to achieve a 10,000-fold amplification RCA-based approaches have recently been attracting attention of diagnostics-oriented biotech companies and research centers for: Gene tests and immunoassays SNP scoring Sequencing template preparation Single-cell analysis systems Gene expression studies 18
- 19.
- 20.
- 21. In 2000, Notomi,T. et al. reported a new method, termed LAMP, that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. 21
- 22. 1. Bst DNApol 2. dNTPs 3. Specific primers (4-6) 4. Target DNA template Use of 4–6 different primers to recognize 6-8 distinct regions The outer primers are known as F3 and B3 inner primers are forward inner primer (FIB) and backward inner primer (BIP) 22
- 23. External primer F3 Internal primer FIP External primerB3 Internal primer BIP Loop primer FL Loop primer BL F3c F2c B2c B3cF3 F2 B2 B3 3’ 5’ 5’ 3’ F1c F2 B2 B1c F1c F1 B1 B1c FL FLc BLc BL Loop primers are complementary to the single stranded loop region. They provide additional starting sites for DNA synthesis and accelerate the amplification thereby reducing the reaction time to less than 30 min. Copyright
- 24.
- 25.
- 26. The mainadvantage of this technique is its simplicity The reaction process proceeds at a constant temperature (60–65 °C) and is completed within 60 min All steps from amplification to detection are conducted within one reaction tube under isothermal conditions prevent contamination 26
- 27. The finalproducts are stem loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops due to hybridization between alternately inverted repeats in the same strand Positive LAMP reactions can be visualized with the naked eye 27
- 28. 1. Isothermal conditionsand no sophisticated equipment required 2. Both amplification and detection can be completed in single step 3. high efficiency 4. Increasing the concentration of pyrophosphate ions 5. Tolerance to some inhibitory 6. There is no need for DNA purification 7. Visual detection of the results 28
- 29. Random hexamerprimer for WGA Hyperbranched amplification of target DNA The yield is less dependent on the amount of input DNA because the reaction is self-limited, the yield will depend on the reaction conditions and amount of reagents and hence on the reaction volume 29
- 30.
- 31. MDA canbe performed directly on crude samples and eliminates the need for an initial DNA purification step Sample with degraded DNA RCA-RCA(restriction and circularization-aided rolling circle amplification) 31
- 32. Replication ofthe NA sequences by multiple primers In one preferred form of the method, two sets of primers are used, a right set and a left set In another preferred form of the method, referred to as whole genome strand displacement amplification, a random set of primers is used to randomly prime a sample of genomic NA The MDA products from a single cell have also been successfully used in array CGH experiments, which usually require a relatively large amount of amplified DNA 32
- 33. 33 Nucleosides, Nucleotides, and NucleicAcids, 27:224–243, 2008
- 34. Initial HAD: 1.Ecoli UvrD 2. T4 SSB 3. Exo-def DNA pol (exo- klenow) Several hundred base pairs of DNA were amplified Kong et al : 1. MutL 2. Ecoli UvrD 3. T4 SSB 4. Exo-def DNA pol 34
- 35. 35 Cell. Mol. LifeSci. (2009) 66:3325–3336
- 36. Tet UvrDfrom Thermoanaerobacter tengcongensis Bacillus stearothermophilus DNA polymerase I large fragment Removing MutL and SSB Amplification at 60–65C 36
- 37. The highprocessivity of the T7 bacteriophage replication system compared with the E. coli UvrD system makes this machinery attractive for long DNA amplification The T7 bacteriophage replisome consists of four proteins phage-encoded gp4 (helicase and primase) gp5 (DNA polymerase) gp2.5 (ssDNA binding protein) host-encoded thioredoxin 37
- 38.
- 39. 39Cell. Mol. LifeSci. (2009) 66:3325–3336
- 40. Using primase-basedwhole genome amplification (pWGA) microgram quantities of DNA product could be obtained from nanogram quantities of input DNA within an hour More importantly, elimination of primer annealing reduces the risk of amplification bias due to uneven annealing of the added primers. 40
- 41. The T7 helicase-primasegp4 denatures the dsDNA template and synthesizes primers Primers are extended by the T7 DNA polymerase gp5/trx complex, resulting in DNA replication in both strands Newly synthesized DNA is displaced and serves as a template for whole genome amplification. 41 Primase-based whole genome amplification
- 42.
- 43. 1. A single,target-specific chimeric primer 2. RNase H 3. DNA pol with a strong strand displacement activity 4. In Ribo-SPIA (for RNA amplification)RT is also added 43
- 44.
- 45. Amplification oflarge populations of NA species, which are limited in biological samples linear and isothermal amplification of the mRNA species in a total RNA population Ribo-SPIA™ is suitable for amplification of mRNA from very small samples (as little as one nanogram total RNA) 45
- 46. 1. Processive T7helicase 2. Exo-def T7 DNA pol (T7 sequenase) 3. T7 Gp2.5 SSB The process can be carried out at one temperature (25◦C) for the entire process Amplification can be performed using purified plasmid DNA or crude cell lysate can amplify inserts as large as 10 Kbps 46
- 47.
- 48.
- 49.
- 50. 1. Kost, G.J. (2002). Goals, guidelines, and principles for point-of-care testing. Kost GJ (ed), 3-12. 2. Lovmar, Lovisa, and Ann‐Christine Syvänen. "Multiple displacement amplification to create a long‐lasting source of DNA for genetic studies." Human mutation 27.7 (2006): 603-614. 3. Hall, M. J., et al. "Use of signal-mediated amplification of RNA technology (SMART) to detect marine cyanophage DNA." Biotechniques 32.3 (2002): 604-611. 4. Jeong, Yong-Joo, Kkothanahreum Park, and Dong-Eun Kim. "Isothermal DNA amplification in vitro: the helicase- dependent amplification system." Cellular and molecular life sciences 66.20 (2009): 3325-3336. 5. Lasken, Roger S. "Single-cell genomic sequencing using multiple displacement amplification." Current opinion in microbiology 10.5 (2007): 510-516. 6. Zhao, Weian, et al. "Rolling circle amplification: applications in nanotechnology and biodetection with functional nucleic acids." Angewandte Chemie International Edition 47.34 (2008): 6330-6337. 7. Gill, Pooria, and Amir Ghaemi. "Nucleic acid isothermal amplification technologies—a review." Nucleosides, Nucleotides, and Nucleic Acids 27.3 (2008): 224-243. 8. Fakruddin, Md, et al. "Nucleic acid amplification: Alternative methods of polymerase chain reaction." Journal of pharmacy & bioallied sciences 5.4 (2013): 245. 9. Polidoros, Alexios N., Konstantinos Pasentsis, and Athanasios S. Tsaftaris. "Rolling circle amplification-RACE: a method for simultaneous isolation of 5'and 3'cDNA ends from amplified cDNA templates." Biotechniques 41.1 (2006): 35. 50
- 51.
- 52. 4 primers(B1,B2, S1, and S2), present in excess, and bind the target strands at positions flanking the sequence to be amplified The 4 primers are simultaneously extended by exo-def klenow using dGTP, dCTP, TTP, and dATP(αS) HincII cuts the hemiphosphorothioate HincII site 52
- 53. RE nicksthe unmodified strand of its target and an exo-def DNA pol extends the 3’ end at the nick and displace the downstream strand BDProbeTec™ ET System: SDA technology has been used mainly for clinical diagnosis of infectious diseases such as chlamydia and gonorrhea SDA products are hybridized with a fluorescent detector probe and are captured by a chemiluminescent assay Greater sensitivity than previous techniques (direct probes, enzyme immunoassays (EIA) and culture) 53
- 54. 54 Nucleosides, Nucleotides, andNucleic Acids, 27:224–243, 2008
- 55. 55 Nucleosides, Nucleotides, andNucleic Acids, 27:224–243, 2008
- 56.
- 57.
- 58.
Editor's Notes
- #15 Two facilitator probes (f1 and f2) anneal to target sequences adjacent to the regions hybridizing to the extension and template probes, which improves the efficiency of three-way junction formation with double-stranded targets
- #23 These advantages can be used to prevent contamination, which can occur in PCR during the transfer of samples containing amplicons from tubes to gels for electrophoretic confirmation Various sizes of loop between F2c and F1c and between B2c and B1c were examined and best results are given when loops of 40 nucleotides (40nt) or longer are used
- #27 The F1c and B1c Tm values should be a little higher than those of F2 and B2 to form the looped out structure. The Tm values of the outer primers F3 and B3 have to be lower than those of F2 and B2 to assure that the inner primers start synthesis earlier than the outer primers. Additionally, the concentrations of the inner primers are higher than the concentrations of the outer primers (Notomi et al. 2000). Furthermore, it is critical
- #28 All four primers are used in the initial steps of the reaction, but in the later cycling steps only the inner primers are used for strand displacement synthesis.
- #29 white precipitate enables visual detection of positive LAMP reactions
- #33 Amplification proceeds by replication with a highly processive polymerase initiated at each primer and continuing until spontaneous termination. In this way, multiple overlapping copies of the entire genome to be synthesized in a short time.
- #38 30 ? 50 exonuclease-deficient T7 DNA polymerase (T7 Sequenase