isothermal nucleic acid amplification techniques
TRANSCRIPT
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Isothermal Nucleic Acid Amplification Techniques
Advisor : Dr. Hossein KhanahmadBy Aref Farrokhi [email protected]
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Terminology
Introduction
Techniques
Conclusion
References
OUTLINE
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TMA : Transcription mediated 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
Terminology and abbreviations
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3WJ : Three-way junction
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
29 Pol : DNA Polymerase from the Bacillus subtilis phage phi29 with exceptional strand displacement and processive synthesis properties and inherent 35' proofreading exonuclease activity
Terminology and abbreviations
SS : single strand
NA : nucleic acid
Pol: polymerase
RE : restriction enzyme
exo-def : exonuclease deficient
Kbps : kilo base pairs
3SR : self-sustained sequence replication
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NA amplification is 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:
High cost of equipment
Contamination chances
Sensitivity to certain classes of contaminants and inhibitors
Can not be use in POCT
Introduction
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RNA pol to 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
NASBA is very similar to TMA
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Gel electrophoresis
Fluorescence probes (real-time NASBA)
Colorimetric assay (NASBA-ELISA)
ECL
Detection of NASBA products
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http://www.premierbiosoft.com/tech_notes/NASBA.html
http://www.bbcorp.co.kr
Nucleic acid sequence based amplification
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The target sequence 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
SMART process is a signal amplification method
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T7 RNA pol 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)
The RNA signal generated may be increased further using additional rounds of extension and transcription
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BioTechniques 32:604-611 (March 2002)
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
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The end detection 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
RNA amplicons can be detected by ELOSA or in real time format
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Phi 29 DNA 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
Rolling circle amplification technology
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Principal mechanism for rolling circle amplification (RCA)
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Ligation reaction of padlock 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
MPRCA uses phi29 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
Recently, RCA has been further developed in a technique, named multiply-primed RCA
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Scheme for multiply-primed rolling circle amplification
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Rapid amplification of cDNA ends (RACE) is the most popular approach for obtaining full-length cDNAs when only part of the transcript's sequence is known
In 2000, Notomi, T. et al. reported a new method, termed LAMP, that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions.
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Bst DNA pol
dNTPs
Specific primers (4-6)
Target DNA template
Use of 46 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)
LAMP reaction
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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
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External primer F3
Internal primer FIP
External primer B3
Internal primer BIP
Loop primer FL
Loop primer BL
F3c F2c
B2c B3c
F3 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
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The main advantage of this technique is its simplicity
The reaction process proceeds at a constant temperature (6065 C) and is completed within 60 min
All steps from amplification to detection are conducted within one reaction tube under isothermal conditions prevent contamination
Only a water bath or heating block is needed to provide a constant temperature
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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
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The final products 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
The final products of LAMP are stem loop DNAs
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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.
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Isothermal conditions and no sophisticated equipment required
Both amplification and detection can be completed in single step
high efficiency
Increasing the concentration of pyrophosphate ions
Tolerance to some inhibitory
There is no need for DNA purification
Visual detection of the results
Advantages of LAMP
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white precipitate enables visual detection of positive LAMP reactions
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Random hexamer primer 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
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Phi 29 is a key element in MDA
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MDA can be 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)
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MDA can be performed directly on crude samples
Replication of the 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
Isothermal multiple displacement amplification
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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.
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Schematic representation of IMDA mechanism
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Nucleosides, Nucleotides, and Nucleic Acids, 27:224243, 2008
Initial HAD:
Ecoli UvrD
T4 SSB
Exo-def DNA pol (exo- klenow)
Several hundred base pairs of DNA were amplified
Kong et al :
MutL
Ecoli UvrD
T4 SSB
Exo-def DNA pol
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HDA: an exponential amplification
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Cell. Mol. Life Sci. (2009) 66:33253336
Tet UvrD from Thermoanaerobacter tengcongensis
Bacillus stearothermophilus DNA polymerase I large fragment
Removing MutL and SSB
Amplification at 6065C
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Kong et al develop a new HDA
The high processivity 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
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T7 replication system for long DNA amplification
30 ? 50 exonuclease-deficient T7
DNA polymerase (T7 Sequenase
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Using primase-based whole 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.
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The T7 DNA pol(gp4) can synthesize DNA by extending from the short RNA primers
The T7 helicase-primase gp4 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.
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Primase-based whole genome amplification
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A single, target-specific chimeric primer
RNase H
DNA pol with a strong strand displacement activity
In Ribo-SPIA (for RNA amplification)RT is also added
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SPIA : Single primer isothermal amplification
Schematic representation of the 3-initiated Ribo-SPIA process
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Amplification of large 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)
Ribo-SPIA :Replication up to 10,000 times off of each original transcript
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Processive T7 helicase
Exo-def T7 DNA pol (T7 sequenase)
T7 Gp2.5 SSB
The process can be carried out at one temperature (25C) for the entire process
Amplification can be performed using purified plasmid DNA or crude cell lysate can amplify inserts as large as 10 Kbps
cHDA is based on the T7 replication machinery
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cHDA mechanism
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CONCLUSION
Summary of isothermal nucleic acid amplification methods
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Kost, G. J. (2002). Goals, guidelines, and principles for point-of-care testing. Kost GJ (ed), 3-12.
Lovmar, Lovisa, and AnnChristine Syvnen. "Multiple displacement amplification to create a longlasting source of DNA for genetic studies." Human mutation 27.7 (2006): 603-614.
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.
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.
Lasken, Roger S. "Single-cell genomic sequencing using multiple displacement amplification."Current opinion in microbiology10.5 (2007): 510-516.
Zhao, Weian, et al. "Rolling circle amplification: applications in nanotechnology and biodetection with functional nucleic acids."Angewandte Chemie International Edition47.34 (2008): 6330-6337.
Gill, Pooria, and Amir Ghaemi. "Nucleic acid isothermal amplification technologiesa review." Nucleosides, Nucleotides, and Nucleic Acids 27.3 (2008): 224-243.
Fakruddin, Md, et al. "Nucleic acid amplification: Alternative methods of polymerase chain reaction." Journal of pharmacy & bioallied sciences 5.4 (2013): 245.
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."Biotechniques41.1 (2006): 35.
References
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Thank you for your atention
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(S)
HincII cuts the hemiphosphorothioate HincII site
SDA uses 4 primers, dATP(S), exo-def klenow and HincII
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RE nicks the 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)
SDA provide exponential amplification of the target DNA
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Nucleosides, Nucleotides, and Nucleic Acids, 27:224243, 2008
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Nucleosides, Nucleotides, and Nucleic Acids, 27:224243, 2008
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Strand Displacement Amplification and Homogeneous Real-Time Detection Incorporated in a Second-Generation DNA Probe System, BDProbeTecET
http://www.clinchem.org/content/45/6/777/F1.expansion?ck=nck
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J Pharm Bioallied Sci. 2013 Oct-Dec; 5(4): 245252