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Isothermal Nucleic Acid Amplification Techniques

Advisor : Dr. Hossein KhanahmadBy Aref Farrokhi Fardaarreeff@ymail.com

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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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Biosensors 2013, 3, 18-43

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

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5

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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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HUMAN MUTATION 27(7),603^614,2006

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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Cell. Mol. Life Sci. (2009) 66:33253336

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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