scholarly journals Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Katharine Best ◽  
Theres Oakes ◽  
James M. Heather ◽  
John Shawe-Taylor ◽  
Benny Chain
Keyword(s):  
Single Molecule ◽  
Computational Analysis ◽  
Pcr Amplification ◽  
Amplification Efficiency ◽  
Stochastic Heterogeneity

scholarly journals Sequence and primer independent stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding

2014 ◽  
Author(s):  
Katharine Best ◽  
Theres Oakes ◽  
James M Heather ◽  
John Shawe-Taylor ◽  
Benny Chain
Keyword(s):  
Single Molecule ◽  
High Throughput Sequencing ◽  
Pcr Amplification ◽  
Transcript Abundance ◽  
Cell Receptor ◽  
Amplification Efficiency ◽  
Experimental Conditions ◽  
Quantitative Results ◽  
Target Molecules ◽  
Stochastic Heterogeneity

The polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology. In combination with High Throughput Sequencing (HTS), PCR is widely used to quantify transcript abundance for RNA-seq and especially in the context of analysis of T cell and B cell receptor repertoires. In this study, we combine molecular DNA barcoding with HTS to quantify PCR output from individual target molecules. Our results demonstrate that the PCR process exhibits very significant unexpected heterogeneity, which is independent of the sequence of the primers or target, and independent of bulk experimental conditions. The mechanistic origin of this heterogeneity is not clear, but simulations suggest that it must derive from inherited differences between different DNA molecules within the reaction. The results illustrate that single molecule barcoding is important in order to derive reproducible quantitative results from any protocol which combines PCR with HTS.

scholarly journals Extreme PCR: Efficient and Specific DNA Amplification in 15–60 Seconds

2015 ◽  
Vol 61 (1) ◽  
pp. 145-153 ◽  
Author(s):  
Jared S Farrar ◽  
Carl T Wittwer
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
High Resolution Melting ◽  
Dna Amplification ◽  
Temperature Cycling ◽  
High Yield ◽  
Temperature Cycle ◽  
Amplification Efficiency ◽  
Analytical Sensitivity ◽  
Agarose Gels

Abstract BACKGROUND PCR is a key technology in molecular biology and diagnostics that typically amplifies and quantifies specific DNA fragments in about an hour. However, the kinetic limits of PCR are unknown. METHODS We developed prototype instruments to temperature cycle 1- to 5-μL samples in 0.4–2.0 s at annealing/extension temperatures of 62 °C–76 °C and denaturation temperatures of 85 °C–92 °C. Primer and polymerase concentrations were increased 10- to 20-fold above typical concentrations to match the kinetics of primer annealing and polymerase extension to the faster temperature cycling. We assessed analytical specificity and yield on agarose gels and by high-resolution melting analysis. Amplification efficiency and analytical sensitivity were demonstrated by real-time optical monitoring. RESULTS Using single-copy genes from human genomic DNA, we amplified 45- to 102-bp targets in 15–60 s. Agarose gels showed bright single bands at the expected size, and high-resolution melting curves revealed single products without using any “hot start” technique. Amplification efficiencies were 91.7%–95.8% by use of 0.8- to 1.9-s cycles with single-molecule sensitivity. A 60-bp genomic target was amplified in 14.7 s by use of 35 cycles. CONCLUSIONS The time required for PCR is inversely related to the concentration of critical reactants. By increasing primer and polymerase concentrations 10- to 20-fold with temperature cycles of 0.4–2.0 s, efficient (>90%), specific, high-yield PCR from human DNA is possible in <15 s. Extreme PCR demonstrates the feasibility of while-you-wait testing for infectious disease, forensics, and any application where immediate results may be critical.

scholarly journals Method Comparison of DNA Isolation and Quantification for Fish and Seafood Authenticity Determination

2018 ◽  
Vol 13 (3) ◽  
pp. 115
Author(s):  
Dwiyitno Dwiyitno ◽  
Stefan Hoffman ◽  
Koen Parmentier ◽  
Chris Van Keer
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Extraction ◽  
Dna Isolation ◽  
Blue Mussel ◽  
Pcr Amplification ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Seafood Products ◽  
Polymerase Chain ◽  
Commercial Kit

Fish and seafood products has been commonly targeted for fraudulent activities. For that reason, authentication of fish and seafood products is important to protect consumers from fraudulent and adulteration practices, as well as to implement traceability regulation. From the viewpoint of food safety, authenticity is beneficial to protect public from serious food poisoning incidents, such as due to ingestion of toxic species. Since DNA based identification depends on the nucleic acid polymerase chain reaction (PCR), the quantity and quality/purity of DNA will contribute significantly to the species authentication. In the present study, different DNA extraction and purification methods (3 classical methods and one commercial kit) were compared to produce the better isolated DNA for PCR amplification. Additionally, different methods for the estimation of DNA concentration and purity which is essential for PCR amplification efficiency were also evaluated. The result showed that classical DNA extraction methods (based on TNES-Urea) yielded a higher amount of DNA (11.30-323.60 ng/g tissue) in comparison to commercial kit/Wizard Promega (5.70-83.45 ng/g tissue). Based on the purity of DNA extract (A260/280), classical DNA extraction method produced relatively similar on DNA quality to the commercial kit (1.79-2.12). Interestingly, all classical methods produced DNA with A260/280 ratio of more than 2.00 on the blue mussel, in contrast with commercial kit. The commercial kit also produced better quality of DNA compared to the classical methods, showing the higher efficiency in PCR amplification. NanoDrop is promising as cheap, robust and safe UV-spectrophotometer method for DNA quantification, as well as the purity evaluation.Keywords: seafood authenticity, DNA isolation, polymerase chain reaction, NanoDrop, Picogreen

scholarly journals Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the ACKR1 Gene Encoding the Duffy Antigens

2019 ◽  
Vol 47 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Yann Fichou ◽  
Isabelle Berlivet ◽  
Gaëlle Richard ◽  
Christophe Tournamille ◽  
Lilian Castilho ◽  
...  
Keyword(s):  
Blood Group ◽  
Single Molecule ◽  
Pcr Amplification ◽  
Null Alleles ◽  
Sequencing Technology ◽  
Gene Encoding ◽  
Next Generation Sequencing Technology ◽  
Sequencing Technologies ◽  
Long Read ◽  
Long Range Pcr

Background: In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of ACKR1, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. Materials and Methods: After long-range PCR amplification spanning the whole ACKR1 gene locus (∼2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. Results: High-quality sequencing reads were obtained for the 162 alleles (accuracy >0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 ACKR1*01, 63 ACKR1*02, and 53 ACKR1*02N.01 alleles, respectively. Discussion: Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a “longitudinal” overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.

scholarly journals pcrEfficiency: a Web tool for PCR amplification efficiency prediction

2011 ◽  
Vol 12 (1) ◽  
Author(s):  
Izaskun Mallona ◽  
Julia Weiss ◽  
Marcos Egea-Cortines
Keyword(s):  
Pcr Amplification ◽  
Amplification Efficiency ◽  
Web Tool

scholarly journals Identification and Prevention of Genotyping Errors Caused by G-Quadruplex– and i-Motif–Like Sequences

2009 ◽  
Vol 55 (7) ◽  
pp. 1361-1371 ◽  
Author(s):  
Jürgen J Wenzel ◽  
Heidi Rossmann ◽  
Christian Fottner ◽  
Stefan Neuwirth ◽  
Carolin Neukirch ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Mendelian Inheritance ◽  
Magnesium Concentration ◽  
Amplification Efficiency ◽  
Nucleotide Polymorphisms ◽  
Genotyping Errors ◽  
Genetic Analyses ◽  
Intronic Polymorphism ◽  
G Quadruplex ◽  
The Impact

Abstract Background: Reliable PCR amplification of DNA fragments is the prerequisite for most genetic assays. We investigated the impact of G-quadruplex– or i-motif–like sequences on the reliability of PCR-based genetic analyses. Methods: We found the sequence context of a common intronic polymorphism in the MEN1 gene (multiple endocrine neoplasia I) to be the cause of systematic genotyping errors by inducing preferential amplification of one allelic variant [allele dropout (ADO)]. Bioinformatic analyses and pyrosequencing-based allele quantification enabled the identification of the underlying DNA structures. Results: We showed that G-quadruplex– or i-motif–like sequences can reproducibly cause ADO. In these cases, amplification efficiency strongly depends on the PCR enzyme and buffer conditions, the magnesium concentration in particular. In a randomly chosen subset of candidate single-nucleotide polymorphisms (SNPs) defined by properties deduced from 2 originally identified ADO cases, we confirmed preferential PCR amplification in up to 50% of the SNPs. We subsequently identified G-quadruplex and i-motifs harboring a SNP that alters the typical motif as the cause of this phenomenon, and a genomewide search based on the respective motifs predicted 0.5% of all SNPs listed by dbSNP and Online Mendelian Inheritance in Man to be potentially affected. Conclusions: Undetected, the described phenomenon produces systematic errors in genetic analyses that may lead to misdiagnoses in clinical settings. PCR products should be checked for G-quadruplex and i-motifs to avoid the formation of ADO-causing secondary structures. Truly affected assays can then be identified by a simple experimental procedure, which simultaneously provides the solution to the problem. .

scholarly journals A New Self-Activated Micropumping Mechanism Capable of Continuous-Flow and Real-Time PCR Amplification Inside 3D Spiral Microreactor

Micromachines ◽  
2019 ◽  
Vol 10 (10) ◽  
pp. 685
Author(s):  
Kangning Wang ◽  
Di Wu ◽  
Wenming Wu
Keyword(s):  
Real Time ◽  
Continuous Flow ◽  
Capillary Tube ◽  
Pcr Amplification ◽  
Amplification Efficiency ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Fluid Resistance ◽  
Long Time ◽  
Quartz Capillary

A self-activated micropump which is capable of stable velocity transport for a liquid to flow a given distance inside a 3D microchannel has been a dream of microfluidic scientists for a long time. A new self-activated pumping mechanism has been proposed in this paper. It is different from the authors’ previous research which relied on the fluid resistance of a quartz capillary tube or end-blocked gas-permeable silicone or a polydimethylsiloxane (PDMS) wall to automate the flow. In this research, an end-open stretched Teflon tube was utilized for passive transport for the first time. A new fluid transmission mode was adopted with the assistance of a cheaper easily accessible oil mixture to achieve stable continuous flow. Finally, this novel micropump has been applied to real-time continuous-flow polymerase chain reactions (PCRs), with an amplification efficiency similar to that of a commercial PCR cycler instrument.

scholarly journals Single Molecule Sequencing of Free DNA from Maternal Plasma for Noninvasive Trisomy 21 Detection

2012 ◽  
Vol 58 (4) ◽  
pp. 699-706 ◽  
Author(s):  
Jessica M E van den Oever ◽  
Sahila Balkassmi ◽  
E Joanne Verweij ◽  
Maarten van Iterson ◽  
Phebe N Adama van Scheltema ◽  
...  
Keyword(s):  
Single Molecule ◽  
Trisomy 21 ◽  
Sequence Data ◽  
Pcr Amplification ◽  
Maternal Plasma ◽  
Single Molecule Sequencing ◽  
Experimental Noise ◽  
Fetal Aneuploidy ◽  
Free Dna ◽  
Gc Bias

Abstract BACKGROUND Noninvasive fetal aneuploidy detection by use of free DNA from maternal plasma has recently been shown to be achievable by whole genome shotgun sequencing. The high-throughput next-generation sequencing platforms previously tested use a PCR step during sample preparation, which results in amplification bias in GC-rich areas of the human genome. To eliminate this bias, and thereby experimental noise, we have used single molecule sequencing as an alternative method. METHODS For noninvasive trisomy 21 detection, we performed single molecule sequencing on the Helicos platform using free DNA isolated from maternal plasma from 9 weeks of gestation onwards. Relative sequence tag density ratios were calculated and results were directly compared to the previously described Illumina GAII platform. RESULTS Sequence data generated without an amplification step show no GC bias. Therefore, with the use of single molecule sequencing all trisomy 21 fetuses could be distinguished more clearly from euploid fetuses. CONCLUSIONS This study shows for the first time that single molecule sequencing is an attractive and easy to use alternative for reliable noninvasive fetal aneuploidy detection in diagnostics. With this approach, previously described experimental noise associated with PCR amplification, such as GC bias, can be overcome.

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