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Isothermal amplification in a lab-on-a-chip

Symposium MFS - Literature thesis
by

Jasper Bouwman

on 11 January 2013

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Transcript of Isothermal amplification in a lab-on-a-chip

Literature thesis Isothermal amplification in a lab-on-a-chip UvA - Literature Thesis MFS
Student:
Supervisors:

Co-assessor:
Date: Human DNA
yes/no The overall picture Part of a larger research

Ultimate goal:
The development of a presumptive human-specific on-chip DNA test

Assessment of the value of a sample at the crime scene


The complete lab-on-a-chip design was beyond the scope of this literature study
Which isothermal amplification techniques are suitable for forensic analysis?


Can these techniques be applied into lab-on-a-chip systems?

Which primers are required to make the test human specific? Research questions Alternative to PCR-based amplification

Amplification at a constant temperature
- enzyme-based

Advantages vs. PCR
- no thermal cycling
- cost effective
- rapid reaction
- simple operation
- less energy required
- more tolerant to inhibitors Isothermal amplification Overwhelming quantity of literature available
Benefit from diagnostics and clinical research




  What is in the literature? Potentially most appropriate methodologies:

Loop-Mediated Isothermal Amplification (LAMP)

Helicase-Dependent Amplification (HDA)

Recombinase Polymerase Amplification (RPA) Results Primer design Primer design Jasper Bouwman
Prof. dr. Han Gardeniers (UT)
Brigitte Bruijns MSc (UT)
Prof. dr. Arian van Asten (NFI)
11-01-2013 The study Overview and thorough review of existing technologies
Suitability conform the forensic field and lab-on-a-chip integration - reaction time
- reaction temperature
- complexity
- specificity
- sensitivity
- tolerance to biological substances
- primer design
- integrated into micro-fluidic device Loop-Mediated Isothermal Amplification (LAMP) Helicase-Dependent Amplification (HDA) Recombinase Polymerase Amplification (RPA) Graduation thesis of Anneloes Bork, University of Twente. Primer design for a presumptive human specific DNA-test, 2012. http://rahadianmf.blogspot.nl/2012/04/photography-birds-eye-view.html (above)
http://www.apple.com/ipad-mini/overview/ (below) http://rt.com/news/sci-tech/dna-fingerprints-fabricated/ Lab-on-a-chip http://www.intellichems.com/Thermometer.html Perform all bio-analytical steps normally conducted in the lab

Potentials:
- hand-held
- cheap
- disposable
- low energy consuming
- small sample size
- low contamination risk

Increasingly being developed http://www.asme.org/kb/news---articles/articles/bioengineering/using-microfluidics-to-diagnose-hiv e.g. immunoglobulin G, heme and heparin http://www.examiner.com/article/what-is-a-research-question 1.



2.

3. There currently exists a diverse range of isothermal amplification technologies each with application specific pros and cons to be considered. Currently available methodologies: among others LAMP, HDA, RCA, SDA, RPA, MDA, NEAR, EXPAR, NASBA, TMA, 3SR, IMDA, SPIA, SMART, SMAP2, ICAN. Selected techniques Applies loop primers to finally create amplicons consisting of multiple stem-loop DNA structures containing several inverted repeats of the target. Most widely researched isothermal amplification technique http://genomicenterprise.com/blog/2010/09/03/peer-review-of-articles-in-science-politically-correct/ Conclusion Recommendations No primers designed due to:
- Time constrains
- Lacking definite choice of isothermal amplification technique

Potential target:
Software:

Human-specific primers for LAMP targeting the cytochrome C gene are already presented in the literature. Extensive research performed concerning the available isothermal amplification technologies

LAMP, HDA and RPA show much potential
Sensitivity of both HDA and RPA is still vague
LAMP satisfies the conditions

Primer design did not succeed due to time constraints
and lacking definite choice of technique
LAMP primers available in literature

A number of recent reports have recommended the need for substantially greater investment into forensic services related research and development Perform additional (literature) research to discover the performance characteristics, with the focus laid on the sensitivity, of both HDA and RPA before continuing with one of these methods.



Pros and cons
Principle LAMP can be seen as a potentially reliable alternative. First test the primers designed by Chu et al, otherwise design them by using the available software to assist. In the meanwhile monitor especially the progress concerning the emerging RPA technique Alu elements
BLAST or websites http://www.sciencedaily.com/releases/2008/05/080526155300.htm http://www.sbm21.com/_r/v.cfm?b=793253&c=17341938 Principle LAMP Tomita, N., et al., Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat Protoc, 2008. 3(5): p. 877-82. Pros and Cons LAMP Pros and Cons HDA Pros and Cons RPA Principle Potentially most appropriate methodologies:

Loop-Mediated Isothermal Amplification (LAMP)

Helicase-Dependent Amplification (HDA)

Recombinase Polymerase Amplification (RPA) Results Potentially most appropriate methodologies:

Loop-Mediated Isothermal Amplification (LAMP)

Helicase-Dependent Amplification (HDA)

Recombinase Polymerase Amplification (RPA) Results Pros and cons Pros and cons Principle HDA Jeong, Y.J., K. Park, and D.E. Kim, Isothermal DNA amplification in vitro: the helicase-dependent amplification system. Cell Mol Life Sci, 2009. 66(20): p. 3325-36. Principle RPA Principle HDA Pros and Cons http://www.twistdx.co.uk/our_technology/ Helicase-Dependent Amplification mimics the natural mechanism of the DNA replication fork.

Understanding this process has recently stimulated the application of helicases for nucleic acid amplification. - Simple technique
- Simple primer design
- Rapid optimization
- Robust to biological substances
- No initial heating step
- Real-time detection
- Integrated in micro-fluidic
device
- Widely researched - Samples containing <100
copies limit the speed
- Indistinct sensitivity
- Expensive enzymes - Highly specific
- Tolerant to biological
substances
- Easy to perform
- Cheap
- Rapid
- Visual + fluorescent detection
- Widely researched

- Complex primer design
- Reaction temperature - Low reaction temperature
- Rapid
- Simple
- Allows 'off-temperature'
- Robust to inhibitory biological
components
- Little in literature
- Incompatible with intercalating
dyes, RT is possible
- Dubious assay performance RPA is a recently developed technology with great potential for implementation into miniaturized devices such as POCT and LOC Characterization of this method is the formation of recombinase filaments - 38% of SC-traces and 17% of HVC-traces resulted in no profile Temperature, polymerase, primers etc. Results Principle
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