40 Inventive Principles for Genetic Diagnostic Laboratories
Supported by grants LO1304, CZ.1.05/3.1.00/14.0307, IGA NT13569, and TE02000058.
Introduction: The Theory of Inventive Problem Solving, TRIZ, is a systematic, knowledge-based procedure for accelerating innovation. The Contradiction Matrix and 40 Inventive Principles are the simplest TRIZ tools that have been used successfully in many technical and non-technical fields; however, TRIZ has not been systematically used to solve genetics diagnostics challenges.
Methods: In this paper I tested by laboratory examination and literature/patent searches whether 40 Inventive Principles were used in molecular genetics laboratories and whether it is possible to derive post hoc Primary Parameter and Conflicting Parameter of the Contradiction Matrix for these Principles.
Results: While I have been able to find working examples for every Inventive Principle in the laboratory, in many instances I have failed to reverse engineer the matching improving and conflicting parameters of the Contradiction Matrix. Therefore, I suggest modifications of the Contradiction Matrix terminology and knowledgebase that would increase the success of its application in the molecular diagnostics analysis domain.
TRIZ, Contradictory matrix, Inventive Principles, molecular genetics
The Theory of Inventive Problem Solving (TRIZ, in Russian language Tеория Решения Изобретательских Задач) is a systematic, knowledge-based procedure for accelerating innovation devised by data mining before the epoch of computers and the big data craze [1,2]. Though it reached popularity in many countries outside Russia, its complexity and lack of standards hinders its universal international acceptance .
The basic TRIZ tool is a collection of 40 Inventive Principles derived in 1956 from patent database searches by Genrich Altshuller. One or more of such Principles is applied to the inventive problem at hand by pre-selection using the Contradiction Matrix. The TRIZ Contradiction Matrix is a two-dimensional table where 39 primary engineering parameters to improve (i.e. Repairability, Reliability, or Measurement accuracy) are positioned in rows while the 39 parameters that can be conflicting are positioned in columns (http://www.triz40.com/TRIZ_GB.php). At the intersection of a given parameter to improve and the undesirable change, the matrix lists the Inventive Principle(s) that apply. For example, the combination of desired Speed and undesired Shape yields the suggestion of three Inventive Principles: Parameter Change, Dynamic Parts, and Vibration. Such repurposing of Inventive Principles to a tangible problem may lead to the generation of inventive ideas in a more structured and efficient way than by using brainstorming or other approaches (brainwriting, mind mapping, lateral thinking, Six Thinking Hats methodology, morphological analysis, etc.) but direct comparison is missing. Indeed, it was applied successfully in diverse contexts, including marketing, psychology, sociology, education, finance, and programming .
Although innovation is clearly a driving force of technological revolutions in genetic diagnostics analysis and TRIZ can facilitate innovation, to my knowledge it has not been systematically applied to solve technical challenges there.
The purpose of this paper is to determine whether the 40 Inventive Principles and 39×39 Contradictory Matrix are applicable to genetic diagnostics. Principles were sought primarily in Polymerase chain reaction (PCR), a basic molecular genetics technique, but the search was open to all molecular genetics techniques, used for diagnosis of the presence of genetic mutation(s).
No systematic review was attempted. Tangible examples of 40 Inventive Principles were found starting with search of the PubMed (http://www.ncbi.nlm.nih.gov/pubmed) database using a combination of title keywords ((PCR [ti] OR polymorphism [ti] OR mutation [ti]) AND (improved OR new OR method OR analysis [ti]) AND genetic* AND diagnostic* AND (Segmentation OR Extraction OR Local quality OR Asymmetry OR Combination OR Universality OR Nesting dolls OR Counterweight OR Prior counteraction OR Prior action OR Cushion OR Equipotentiality OR Inversion OR Spheroid* OR curvature OR Dynamism OR Partial OR excessive OR new dimension OR Mechanical vibration OR Periodic action OR Continuity OR Skipping OR rushing through OR Blessing in disguise OR Feedback OR Intermediary OR Self-service OR Copying OR inexpensive OR disposables OR Replacement OR Pneumat* OR hydraulics OR Flexible films OR membranes OR Porous OR Changing colour OR Homogeneity OR Recycling OR discarding OR regenerating OR Transforming OR Phase transition OR Thermal expansion OR oxidat* OR Inert OR Composite)”. The resulting list of papers was manually pruned according to titles that did not suggest an innovative methodological approach. Abstracts of remaining papers were read and when relevant, Inventive Principles were extracted by reading the whole paper.. Also, the US Patent & Trademark Office (USPTO) (http://patft.uspto.gov/netahtml/PTO/search-adv.htm) was sought for the genetics diagnostics related patent using similar strategy. The number of the followed inventions was reduced to keep the number of references below 100.
Then, occurrences of the Inventive Principles in the Contradiction Matrix and in my screening results were counted (using Python script on the original Contradictory Matrix at http://www.triz-journal.com/contradiction-matrix/ and manually, respectively) and one exemplar problem for each Inventive Principle was chosen for reverse engineering, which was used to determinethe desired and undesired property (Suppl. 1).
1,062 papers were found using the above combination of keywords in the PubMed search. That number was manually reduced to 141 papers upon reading the titles. The list was further shortened and some papers replaced and supplemented with 20 patents found in the USPTO search.
Below are the found applications of the Inventive Principles from screening the genetic diagnostics field. An elaboration of one example for each Principle is in Suppl. 1.
Distance learning, virtual offices; modular laboratories (pre-PCR, PCR, and post-PCR rooms); fragmentation by sonication during next-generation sequencing, shotgun sequencing , independent blocks in PCR thermocycler.
Placing noisy centrifuge in corridor; extracting DNA from samples while omitting proteins, sugars, lipids, and metabolites; switching off pre-PCR polymerase activity by omitting magnesium from reagents cocktail until the last pipetting step ; adding blocking oligonucleotide to prevent non-specific primer binding in PCR [8,9].
3. Local quality
Assigning technicians to specific processes; exploiting specific qualities of restriction and primer binding sites to achieve specificity of genotyping; use of temperature gradient PCR to find the optimum annealing temperature; use of temperature gradient electrophoresis to increase method´s discrimination power ; closed tube nested PCR with second set of reagents in a hanging gel matrix to avoid opening PCR tubes before starting the second, nested reaction .
Use of asymmetric DNA length distribution to authenticate ancient DNA ; asymmetric PCR to amplify exponentially just one template strand .
Parallel cloud computing; Selective Nucleic Acid Removal via Exclusion to simultaneously purify mRNA and DNA ; multiplexing using tagged PCR primers ; combined reverse transcription and PCR in closed tubes .
Printer/scanner/copier/fax machines; tubes suitable both for thermocyclers and fluorimeters; universal PCR primers for tagged amplicons ; peptide nucleic acid oligonucleotides acting as both PCR clamps and sensor probes .
7. Nesting dolls
Minifuge using rotor attachment as vortex; heated electromagnetic mixers; DNA extraction column placed in a 1.5 ml tube; nested PCR .
Gel plugs in centrifuge tubes for separating phases during DNA extraction; excessive primers for long amplicons in multiplex for Short Tandem Repeats (STR) to reverse effect of DNA degradation on fluorescence signal (GlobalFiler, Life Technologies).
9. Prior counteraction
Use of prefilter before HEPA filter in flowbox to increase lifetime of HEPA filter; buffer solution to prevent harm from pH extremes; Taq polymerase with postponed function (HotStart) to reduce unspecific PCR priming , blocking wildtype allele primers to increase PCR sensitivity to mutant alleles ; use of BSA to bind inhibitors in PCR [20,91].
10. Prior action
DNA decontamination from working surfaces using vitamins, metal ion and surface active compound ; checking reagent availability before starting SOP; heatblock pre-heating in anticipation of sample incubation.
11. Cushion in advance
Computer recycle bin allowing undelete; UPS for cycler and PC; antivirus software; dividing expensive solution into aliquots to reduce losses if contamination occurs; incorporating dU into PCR amplicon to ease postPCR decontamination by uracil N-glycosylase .
Movement of pipetting parts instead of sample tubes in DNA extraction automats; touchDown PCR to level out hybridizing requirements of several primer pairs in PCR to achieve specificity with increased yield .
Dispensing nanoliters of solution upwards acoustically; use of short non-specific primers for whole genome amplification; inversion PCR  with 3´ends of primers facing outwards; padlock probes amplifying signals instead of target sequences .
14. Spheroidality or curvature
Use of undulating orbital shaker ; centrifugation during DNA extraction; curvature analysis for fluorescence threshold determination instead of simple fluorescence measurement during quantitative PCR ; PCR tube capping aid with arc shape to maximize pressure on individual caps (Eppendorf, Fisher Scientific).
Dynamic software user interfaces hiding rarely used functions and highlighting next anticipated step; internal length standards for electrophoresis , internal PCR standards in every PCR tube allowing adjustment for uncontrollable factors; dynamic heated lids in PCR cyclers to avoid condensation of PCR mixture on tube cap.
16. Partial or excessive action
Estimating allele frequencies in a population from samples, reducing denaturation temperatures in COLD-PCR to increase detection limit for minority DNA sequences .
17. Another dimension
Use of unnatural nucleotide bases in PCR ; emulsion and digital PCR [31,32,33]; nucleic acid amplification at room temperature using PCR alternatives [34,35], microfluidic droplet technology .
18. Mechanical vibration
Sonication for DNA extraction or for instrument cleaning ; vortexers ; bead beaters ; pressure cycling instruments; bone shredders to help release DNA from hard samples; electrokinetic injection of DNA fragments for capillary electrophoresis; vibrating PCR tubes to launch second round PCR in nested PCR .
19. Periodic action
Shakers for DNA extraction; pulsed field electrophoresis to increase discrimination power for long stretches of DNA ; flashing light controls in PCR machines to indicate runs in progress; PCR per se .
20. Continuity of useful action
Simultaneous running two experiments (e.g. preparing one set of samples while another is in a thermocycler); automated overnight sample loading; pumping reaction mixtures through different temperature zones in PCR cycling [43,44].
21. Rushing through
Laser capture microdissection; fast PCR [45,46].
22. Blessing in disguise
Intended mismatch increasing specificity of PCR , combined use of proofreading and non-proofreading polymerases for long PCR .
Calibration, optimization, and accreditation processes in the laboratory; use of cresol red as inert pipetting aid for PCR mixtures . In situ monitoring of extension rates during realtime PCR .
Use of gel plug during nucleic acid extraction with organic chemicals , reverse (RNA to cDNA) transcription for PCR; BSA to sequester Taq polymerase inhibitors .
Autoclavable pipettes; wax PCR overlay to prevent contamination after PCR ; inorganic phosphate release upon dNTP incorporation into nascent DNA strands for signal detection during pyrosequencing .
Software tutorials and virtual reality; mock samples made by genetic engineering for training and method validation (Horizon Diagnostics reference standards); use of whole genome amplified DNA instead of original human sample with mutation for positive PCR control; analysis of PCR amplicons rather than gDNA .
27. Cheap disposables
Disposable pipette tips instead of disposable pipettes, laboratory coats, gloves, and slippers; portable nucleic acid extraction system using bicycle pump ; degradable primers .
28. Replacement of mechanical systems
Use of calibrated pipette tips instead of weighing liquid drops for pipette calibration ; heated air in PCR thermocycler instead of Peltier pump .
29. Pneumatics or hydraulics
Use of vacuum instead of centrifugation for moving DNA eluates into archiving tube; integrated pneumatic micropumps for droplet generation in lab-on-chip  or PCR .
30. Flexible films or membranes
Parafilm for dispensing agarose electrophoresis loading buffer; membrane columns for DNA extraction; rubber cover or film for multi-PCR tube plates. Membrane arrays for SNP genotyping ; surface passivation of PCR chips by silane treatment .
31. Porous materials
Membrane columns for sterilizing solutions or extracting DNA; sieving electrophoresis polymers to increase discrimination power; buccal swab brushes to maximize yield of trace DNA collection ; microfluidic PCR tube for fast PCR (SmartCycler, Cepheid).
32. Changing color
pH color indicators during DNA extraction; use of fluorescent resonance energy transfer (FRET) in oligonucleotide probes ; fluorescence tagging of primers for capillary electrophoresis with laser detection; cresol red as color indicator during premixing of PCR reagents and electrophoresis sample loading .
Closed-tube (homogenous) SNP detection using FRET ; isothermal amplification and HyBeacon probes ; detection of proteins by assembling DNA.
34. Discarding, recycling, and regenerating
Pass amplicon samples through columns for getting rid of unused PCR reagents; hanging mercury drop electrode for nucleic acid detection ; wax capsules with magnesium and polymerase for PCR; wax layer barrier to sequester Phi29 pre-amplification from a target-specific real-time PCR reaction .
35. Transforming physical or chemical properties
Use of pressure cooker or microwave for dissolving agarose; paraffin-, chitosan-, trehalose- or pullulan-encapsulation for archiving of DNA or PCR reagents .
36. Phase transition
Evaporating residual ethanol from precipitates before dissolving DNA; use of low melting temperature agarose to ease nucleic acid manipulation after electrophoresis ; freezing dissolved DNA or pipetting it on FTA card for long-term storage; dry PCR reagents .
37. Thermal expansion
Film for closing PCR tubes by heating instead of caps; high pH PCR buffer to control pH reductions in high temperature PCR steps and reduce effects of contaminants in whole blood samples .
38. Strong oxidants
Use of oxidation of luciferin for detection during pyrosequencing ; use of oxidation of guanine on biosensors ; PCR decontamination by bleach .
39. Inert environment
Mineral oil overlay of PCR reagents; DNA/pyrogen/DNase/metal-free disposables; DNA encapsulation in magnetically recoverable, thermostable, hydrophobic silica  or agarose sol-gel droplets ; aminoacid backbone of peptide nucleic acid to hide sequence before nucleases .
40. Composite materials
Two filter layers in pipette tips to increase protection; Twin.tec PCR plates to increase resistance to temperature changes ; composite gel to improve resolution of STR alleles in polyacrylamide electrophoresis ; multiplex PCR.
In summary, two to five examples of the application of each Inventive Principle were found in molecular genetics laboratories. In the original Contradictory Matrix, the most frequently used was Principle 35, Transforming physical or chemical properties, which was encountered 411 times, while Principle 20, Continuity of action, was found only 19 times. The difference in relative frequency of Principles in two databases does not justify any conclusion because of the unrepresentativeness of my virtual database.
One of the molecular biology examples for each Principle was analyzed further to test applicability of the Contradiction Matrix, as listed in Suppl. 1.
For Principles 1-6, 17, and 18, the desired and undesired property from 39*39 Contradictory Matrix was found without hesitation. However, for Principles 7-16, 19-40, the search for abstractization of conflicting properties was given up after realizing that it would require more inventive power than looking for the solution to the challenge itself. Rather, a new set of seventeen conflicting parameters was appended into Suppl. 1: pre-analytical requirements, specificity, sensitivity, yield, convenience, price, reproducibility, robustness, turn-around time, real-time monitoring and real-time parameter adjustment, point-of-care testing, shelf life, multi-targeting, automation, decontamination, and timing.
The search results show that all of the 40 TRIZ Principles have already been used in genetic diagnostic laboratories worldwide. In some cases (especially in massively parallel sequencing technologies), numerous principles were used to solve one complicated problem.
In addition, some inventive authors, e.g. Wittwer [58,19,81,82,83,84,85,86,87,88], appear to have used virtually all of Altshuller´s Principles, and it would be interesting to know if they used a systematic innovation approach.
While it was easy for me to find in the genetics laboratories examples of Principle of Segmentation (1), Cushion in advance (11), Mechanical vibration (18), Membranes (30), it was harder to come across examples of Counterweight (8), Equipotentiality (12), Partial or excessive action (16), Skipping (21), Replacement of mechanical system (28), Pneumatics or hydraulics (29), Transformation (35), and Thermal expansion (37). This difference may reflect the subjectivity of my search, and more experienced inventors may achieve a different frequency distribution. However, an uneven number of occurrences in the original Contradiction Matrix (Suppl. 1) shows us that the frequency of some principles was higher even in the original Altshuller database.
It is possible that molecular genetics laboratory challenges are too complicated for the Contradictory Matrix and higher order instruments of TRIZ should be used to provide “multismart” solutions for multifaceted conflicting requirements. Also, molecular genetics and genetic diagnostics are closer to analytical chemistry with a different set of method parameters than the original 39 engineering parameters. Seventeen of the new parameters are listed in the Results but this list should not be considered final due to the limitation of their derivation from one example per each Principle. Thus, it is possible that data mining focused to solutions of genetic diagnostics problems would provide a different Contradiction Matrix. Expansion and update of the Contradiction Matrix for engineers was already performed by Mann . A similar endeavor for molecular genetics would require extraction of semantic information from patent databases and PubMed using software means that were not available to the author at the time of paper writing and were beyond the scope of this article.u
- All 40 TRIZ Inventive Principles have found application in molecular genetics laboratories.
- It was not possible for me to find conflicting parameters from the original Contradiction Matrix for all these Principles. Matrix does not fit perfectly to genetic diagnostics field.
- Contradiction Matrix requires adaptation and update. One possible way of Matrix reconstruction is the use of terms from analytical chemistry as conflicting parameters.
- Despite the lack of literature describing the use of TRIZ techniques in genetics diagnostics, this paper indicates that it is worth further testing at least.
Supported by grants LO1304, CZ.1.05/3.1.00/14.0307, IGA NT13569, and TE02000058.
I thank Petr Vojta, MSc. for extracting information from Contradictory Matrix using Python script and Jana Stránská, PhD. for helpful suggestions regarding improvements of the manuscript. Parts of this paper were presented in a poster at the Forensica 2014 conference in Prague (http://www.forensica2014.org/). I declare no competing interest.
- Altshuller G. Creativity as an Exact Science. CRC Press,1984
One of the important books of the original TRIZ author.
- Kurgan LA, Musilek P. A survey of knowledge discovery and data mining process models. Knowledge Engineering Review 2006;21(1):1-24
- Ilevbare IM, Probert D, Phaal R. A review of TRIZ, and its benefits and challenges in practice. Technovation 2013;33(2-3):30-37
Unbiased, critical review of TRIZ.
- Altshuller G, Shulyak L, Rodman S. 40 Principles: TRIZ Keys to Technical Innovation. Technical Innovation Ctr,2001
Illustrated 40 Inventive Principles.
- Moehrle MG. What is TRIZ? From conceptual basics to a framework for research. CAIM 2005;14(1):3-13
- Favello A, Hillier L, Wilson RK. Genomic DNA sequencing methods. Methods Cell Biol 1995;48:551-569
- Ankenbauer W, Heindl D, Laue F. PCR hot start by magnesium sequestration. US 8,026,058 (2014)
- Makrigiorgos G. Full cold-PCR enrichment with reference blocking sequence. US 8,623,603 (2014)
- Lee S-T, Ki C-S, Kim J-W. Method for detecting genetic mutation by using a blocking primer. US 2013/0149695 A1 (2013)
- Henco K, Heibey M. Quantitative PCR: the determination of template copy numbers by temperature gradient gel electrophoresis (TGGE). Nucleic Acids Res 1990;18(22):6733-6734
- Yourno J. A method for nested PCR with single closed reaction tubes. Genome Res 1992;2(1):60-65
- Malmstrom H, Svensson EM, Gilbert MTP et al. More on contamination: The use of asymmetric molecular behavior to identify authentic ancient human DNA. Mol Biol Evol 2007;24(4):998-1004
- Zhang Z, Wang C, Zhu L et al. Asymmetric PCR amplification, its special primer and application. 8,735,067 (2014)
- Strotman L, O’Connell R, Casavant BP et al. Selective nucleic acid removal via exclusion (SNARE): capturing mRNA and DNA from a single sample. Anal Chem 2013;85(20):9764-9770
- Shuber AP, Grondin VJ, Klinger KW. A simplified procedure for developing multiplex PCRs. Genome Res 1995;5(5):488-493
- Ankenbauer W, Grepl U, Haerteis R. Rapid one-step reverse transcriptase PCR. US 8,119,353 (2014)
- Chiou CC, Luo JD. Methods and kits for the detection of nucleotide mutations using peptide nucleic acid as both PCR clamp and sensor probe. US 7,803,543 (2014)
- Nilsson J, Bosnes M, Larsen F et al. Heat-mediated activation of affinity-immobilized Taq DNA polymerase. Biotechniques 1997;22(4):744-751
- Smith GD, Zhou L, Rowe LR et al. Allele-Specific PCR with competitive probe blocking for sensitive and specific detection of BRAF V600E in thyroid fine-needle aspiration specimens. Acta Cytol 2011;55(6):576-583
- Okajima H, Yamamoto T, Kojima T et al. Amplification of HLA-DQA1 gene from bloodstains by polymerase chain reaction. Nihon Hoigaku Zasshi 1993;47(1):6-12
- Esser KH, Marx WH, Lisowsky T. DNA decontamination: DNA-ExitusPlus in comparison with conventional reagents. Nat Methods 2006;3(2):I-II
- Rys PN, Persing DH. Preventing false positives: quantitative evaluation of three protocols for inactivation of polymerase chain reaction amplification products. J Clin Microbiol 1993;31(9):2356-2360
- Don RH, Cox PT, Wainwright BJ et al. ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 1991;19(14):4008-
- Ochman H, Gerber AS, Hartl DL. Genetic applications of an inverse polymerase chain reaction. Genetics 1988;120(3):621-623
- Nilsson M, Malmgren H, Samiotaki M et al. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 1994;265(5181):2085-2088
- Reynolds CS, Reynolds Jr, Albert B et al. High/low profile rocker. US 5,423,603 (1995)
- Kurnik RT, Wang J. PCR elbow determination using curvature analysis of a double sigmoid. US 7991562 B2 (2011)
- Simons MJ. Internal standard for electrophoretic separations. US 5,096,557 (1992)
- Li J, Wang L, Mamon H et al. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 2008;14(5):579-584
- Moser MJ, Marshall DJ, Grenier JK et al. Exploiting the enzymatic recognition of an unnatural base pair to develop a universal genetic analysis system. Clin Chem 2003;49(3):407-414
- Nakano M, Komatsu J, Matsuura S et al. Single-molecule PCR using water-in-oil emulsion. J Biotechnol 2003;102(2):117-124
- Lo YMD, Chiu RWK. Analysis for nucleic acids by digital PCR . US 8,722,334 (2014)
- Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci U S A-Biol Sci 1999;96(16):9236-9241
- Fakruddin M, Mannan KS, Chowdhury A et al. Nucleic acid amplification: Alternative methods of polymerase chain reaction. J Pharm Bioallied Sci 2013;5(4):245-252
Short review of PCR alternatives.
- Sidoti F, Bergallo M, Costa C et al. Alternative molecular tests for virological diagnosis. Mol Biotechnol 2013;53(3):352-362
- Zec H, Shin DJ, Wang TH. Novel droplet platforms for the detection of disease biomarkers. Expert Rev Mol Diagn 2014;14(7):787-801
- Heller MJ, Robinson RA, Burgart LJ et al. DNA extraction by sonication: a comparison of fresh, frozen, and paraffin-embedded tissues extracted for use in polymerase chain reaction assays. Mod Pathol 1992;5(2):203-206
- Gebrian PL. Method and apparatus for mixing liquid solutions using a rotating magnet to generate a stirring vortex action. US 6,382,827 (2002)
- Hwang KY, Kwon SH, Jung SO et al. Miniaturized bead-beating device to automate full DNA sample preparation processes for gram-positive bacteria. Lab Chip 2011;11(21):3649-3655
- Cheng J, Guo M, Jiang D et al. A kind of net PCR reaction tube. WO 2006050636 A1 (2006)
- Schwartz DC, Cantor CR. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 1984;37(1):67-75
- Mullis K, Faloona F, Scharf S et al. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol 1986;51 Pt 1:263-273
- Nakano H, Matsuda K, Yohda M et al. High speed polymerase chain reaction in constant flow. Biosci Biotechnol Biochem 1994;58(2):349-352
- Stanley K, Corbett J. Thermocycler and sample port. US 8,124,413 (2012)
- Wittwer CT, Fillmore GC, Garling DJ. Minimizing the time required for DNA amplification by efficient heat transfer to small samples. Anal Biochem 1990;186(2):328-331
- Wang D, Hennessy L. Fast PCR for STR genotyping. US 8,580,505 (2014)
- Kwok S, Chang SY, Sninsky JJ et al. A guide to the design and use of mismatched and degenerate primers. PCR Methods Appl 1994;3(4):S39-S47
- Cheng S, Fockler C, Barnes WM et al. Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci U S A 1994;91(12):5695-5699
- Hodges E, Boddy SM, Thomas S et al. Modification of IgH PCR clonal analysis by the addition of sucrose and cresol red directly to PCR reaction mixes. J Clin Pathol -Mol Pa 1997;50(3):164-166
- Mondal S, Venkataraman V. In situ monitoring of polymerase extension rate and adaptive feedback control of PCR by using fluorescence measurements. J Biochem Biophys Methods 2005;65(2-3):97-105
- Murphy NR, Hellwig RJ. Improved nucleic acid organic extraction through use of a unique gel barrier material. Biotechniques 1996;21(5):934-939
- Kaijalainen S, Karhunen PJ, Lalu K et al. An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose. Nucleic Acids Res 1993;21(12):2959-2960
- Ahmadian A, Gharizadeh B, Gustafsson AC et al. Single-nucleotide polymorphism analysis by pyrosequencing. Anal Biochem 2000;280(1):103-110
- Mullis KB, Erlich HA, Arnheim N et al. Process for amplifying, detecting, and/or-cloning nucleic acid sequences. US 4,683,195 (1987)
- Byrnes S, Fan A, Trueb J et al. A portable, pressure driven, room temperature nucleic acid extraction and storage system for point of care molecular diagnostics. Anal Methods 2013;5(13):3177-3184
- Du Breuil L, Rusla M. Nested PCR employing degradable primers. US 7,273,730 (2014)
- DeVaughn DH. Calibrated pipette tip and method. US 5,260,030 (1993)
- Wittwer CT, Fillmore GC, Hillyard DR. Automated polymerase chain reaction in capillary tubes with hot air. Nucleic Acids Res 1989;17(11):4353-4357
- Zeng Y, Shin M, Wang T. Programmable active droplet generation enabled by integrated pneumatic micropumps. Lab Chip 2013;13(2):267-273
- Choi JY, Kim YT, Byun JY et al. An integrated allele-specific polymerase chain reaction-microarray chip for multiplex single nucleotide polymorphism typing. Lab Chip 2012;12(24):5146-5154
- Jobs M, Howell WM, Stromqvist L et al. DASH-2: flexible, low-cost, and high-throughput SNP genotyping by dynamic allele-specific hybridization on membrane arrays. Genome Res 2003;13(5):916-924
- Shoffner MA, Cheng J, Hvichia GE et al. Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR. Nucleic Acids Res 1996;24(2):375-379
- Triva D. Method of using flocked swab for collecting biological specimens. US 8,317,728 (2012)
- Chen X, Livak KJ, Kwok PY. A homogeneous, ligase-mediated DNA diagnostic test. Genome Res 1998;8(5):549-556
- Howard RL, French DJ, Richardson JA et al. Rapid detection of diagnostic targets using isothermal amplification and HyBeacon probes–a homogenous system for sequence-specific detection. Mol Cell Probes 2015;29(2):92-98
- Zhang H, Li F, Li XF et al. Yoctomole detection of proteins using solid phase binding-induced DNA assembly. Methods 2013;64(3):322-330
- Huska D, Hubalek J, Adam V et al. Miniaturized electrochemical detector as a tool for detection of DNA amplified by PCR. Electrophoresis 2008;29(24):4964-4971
- Erlandsson L, Nielsen LP, Fomsgaard A. Amp-PCR: combining a random unbiased Phi29-amplification with a specific real-time PCR, performed in one tube to increase PCR sensitivity. P ONE 2010;5(12):e15719-
- Jahanshahi-Anbuhi S, Pennings K, Leung V et al. Pullulan encapsulation of labile biomolecules to give stable bioassay tablets. Angew Chem 2014;53(24):6155-6158
- Burns DM, Beacham IR. A method for the ligation of DNA following isolation from low melting temperature agarose. Anal Biochem 1983;135(1):48-51
- Gumbrecht W, Paulicka P, Stanzel M. PCR process and arrangement for DNA amplification using dry reagents. US 7,993,828 (2014)
- Bu Y, Huang H, Zhou G. Direct polymerase chain reaction (PCR) from human whole blood and filter-paper-dried blood by using a PCR buffer with a higher pH. Anal Biochem 2008;375(2):370-372
- Ahmadian A, Lundeberg J, Nyren P et al. Analysis of the p53 tumor suppressor gene by pyrosequencing. Biotechniques 2000;28(1):140-147
- Tansil NC, Xie F, Xie H et al. An ultrasensitive nucleic acid biosensor based on the catalytic oxidation of guanine by a novel redox threading intercalator. Chem Commun 2005;(8):1064-1066
- Prince AM, Andrus L. PCR: how to kill unwanted DNA. Biotechniques 1992;12(3):358-360
- Puddu M, Paunescu D, Stark WJ et al. Magnetically recoverable, thermostable, hydrophobic DNA/silica encapsulates and their application as invisible oil tags. ACS Nano 2014;8(3):2677-2685
- Leng X, Zhang W, Wang C et al. Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR. Lab Chip 2010;10(21):2841-2843
- Nielsen PE, Egholm M, Buchardt O. Peptide nucleic acid (PNA). A DNA mimic with a peptide backbone. Bioconjug Chem 1994;5(1):3-7
- Turner BR, Titus DE. Thin-well microplate and methods of making same. US 6,340,589 (2002)
- Lorente M, Lorente JA, Wilson MR et al. Composite PAGE: an alternate method for increased separation of amplified short tandem repeat alleles. Int J Legal Med 1993;106(2):69-73
- von Ahsen N, Wittwer CT, Schutz E. Monovalent and divalent salt correction algorithms for Tm prediction–recommendations for Primer3 usage. Brief Bioinform 2011;12(5):514-517
- Seipp MT, Herrmann M, Wittwer CT. Automated DNA extraction, quantification, dilution, and PCR preparation for genotyping by high-resolution melting. J Biomol Tech 2010;21(4):163-166
- Sundberg SO, Wittwer CT, Gao C et al. Spinning disk platform for microfluidic digital Polymerase Chain Reaction. Anal Chem 2010;82(4):1546-1550
- Willmore-Payne C, Holden JA, Wittwer CT et al. The use of EGFR exon 19 and 21 unlabeled DNA probes to screen for activating mutations in non-small cell lung cancer. J Biomol Tech 2008;19(3):217-224
- Margraf RL, Mao R, Wittwer CT. Masking selected sequence variation by incorporating mismatches into melting analysis probes. Hum Mut 2006;27(3):269-278
- Elenitoba-Johnson O, David D, Crews N et al. Plastic versus glass capillaries for rapid-cycle PCR. Biotechniques 2008;44(4):487-8, 490, 492
- Chou LS, Meadows C, Wittwer CT et al. Unlabeled oligonucleotide probes modified with locked nucleic acids for improved mismatch discrimination in genotyping by melting analysis. Biotechniques 2005;39(5):644-648
- Crockett AO, Wittwer CT. Fluorescein-labeled oligonucleotides for real-time pcr: using the inherent quenching of deoxyguanosine nucleotides. Anal Biochem 2001;290(1):89-97
- Mann D, Dewulf B. Matrix 2003 : Updating the TRIZ Contradiction Matrix. CREAX Press;2003
- Porter-Jordan K, Rosenberg EI, Keiser JF et al. Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA. J Med Virol 1990;30(2):85-91
- Olive DM, Simsek M, Al-Mufti S. Polymerase chain reaction assay for detection of human cytomegalovirus. J Clin Microbiol 1989;27(6):1238-1242
- Mayrand PE. Standards and controls in DNA assays. Ann Biol Clin -Paris 1992;50(10-11):719-721
- Eglen RM, Reisine T, Roby P et al. The use of AlphaScreen technology in HTS: current status. Current Chem Genom 2008;1:2-10