Caister Academic Press

Figures from the book: PCR Troubleshooting and Optimization

Pictures and illustrations of PCR and related topics.

Chapter 1: Magic in Solution: An Introduction and Brief History of PCR

Chapter 1. Figure 1. The PCR cycle.
Chapter 1. Figure 2. Cross-sectional diagram of a rapid, air-controlled thermal cycler. An Idaho Technology ATC 1605 thermal cycler from 1990 is shown.
Chapter 1. Figure 3. Equilibrium and kinetic paradigms of PCR.
Chapter 1. Figure 4. Schematic diagram of the academic LightCycler prototype.
Chapter 1. Figure 5. The Idaho Technology LC 24 was the first commercial carousel LightCycler, first sold in 1996.
Chapter 1. Figure 6. The Roche carousel LightCycler (right) and the ABI 7700 (left) were the first two real-time instruments marketed worldwide.
Chapter 1. Figure 7. Continuous monitoring of rapid cycle PCR.
Chapter 1. Figure 8. Rapid-cycle, real-time PCR and melting analysis.

Chapter 2: Difficult Templates and Inhibitors of PCR

Chapter 2. Figure 1. Amplification curves and amplification analysis.
Chapter 2. Figure 2. A Stock I dilution series exposing the "inhibitory characteristic" for a group of sheep total lung RNA samples isolated by TRIzol.
Chapter 2. Figure 3. The "blanking buffer" for nucleic acid quantification.
Chapter 2. Figure 4. Example of a SYBR-Green based assay using P-Q to establish the good, valid dynamic dilution range for samples and standards.
Chapter 2. Figure 5. A partial glimpse of the Questionnaire interface for the PREXCEL-Q program.
Chapter 2. Figure 6. Comparison of one-step SYBR Green-based qPCR master mix from company A and company B on RimM expression profiles from 48 hour-old bacterial bio-film cells.
Chapter 2. Figure 7. Example of the dramatic effect of one-step master mix choice on qPCR performance.

Chapter 3: Significance of Controls and Standard Curves in PCR

Chapter 3. Figure 1. A screenshot from BioRad CFX software showing example of a good standard curve.
Chapter 3. Figure 2. A screenshot from BioRad CFX software showing example of a bad standard curve.

Chapter 4: Obtaining Maximum PCR Sensitivity and Specificity

Chapter 4. Figure 1. Schematic representing key considerations of PCR assay design and optimization.
Chapter 4. Figure 2. Amplification curves on a LightCycler 1.5, in channel 1.
Chapter 4. Figure 3. UCSC in silico amplicon predicts one PCR product.
Chapter 4. Figure 4. Results from UCSC in silico PCR tool showing dual priming to a desired and an undesired site.
Chapter 4. Figure 5. Amplification curves of two hydrolysis probe targets.
Chapter 4. Figure 6. Effect of dNTP variation on qPCR.
Chapter 4. Figure 7. Melting curves and normalized derivative melting peaks showing the difference that primer purification can make.
Chapter 4. Figure 8. Gradient annealing optimization.

Chapter 5: RT-PCR Optimization Strategies

Chapter 5. Figure 1. Example of hook effect in sample with highest starting amount.
Chapter 5. Figure 2. LNA probe with "locked" ring (red lines).

Chapter 6: Real-Time PCR Instrumentation: An Instrument Selection Guide

Chapter 6. Figure 1. The Peltier effect corresponds to the heat transfer from one end of a conductor to the other caused by the passage of an electrical current.
Chapter 6. Figure 2. Emission spectra, and spectral overlap, of four commonly used fluorescent dyes (FAM, HEX, ROX, and Cy5).
Chapter 6. Figure 3. Selection of a qPCR instrument based on throughput, multiplexing technology, and HRM capabilities.
Chapter 6. Figure 4. Images of real-time PCR instruments. Front views of the various real-time PCR instruments are shown, to scale relative to each other.

Chapter 7: qPCR Data Analysis: Unlocking the Secret to Successful Results

Chapter 7. Figure 1. Melt curve analysis.
Chapter 7. Figure 2. Typical standard curve obtained from a 6-point 4-fold dilution series for an assay with a good amplification efficiency.
Chapter 7. Figure 3. Outcome of a geNorm analysis on a heterogeneous cancer biopsy sample set.
Chapter 7. Figure 4. Calculation workflow.
Chapter 7. Figure 5. NRQ value of a target gene for 3 samples in linear scale.

Chapter 8: The MIQE Guidelines Uncloaked

Chapter 8. Table 1. MIQE checklist for authors, reviewers, and editors."

Chapter 9: PCR Applications for Epigenetics Research

Chapter 9. Figure 1. MeDIP and MethylMiner-based methylated DNA enrichment workflows.
Chapter 9. Figure 2. Methylation in the promoter and 5' UTR of the PLAU gene.
Chapter 9. Figure 3. qPCR data from non-enriched DNA (input) is used to evaluate amplicon-specific sequence enrichment.
Chapter 9. Figure 4. Relative enrichment analyses of MethylMiner-based separation of non-methylated and methylated sequences.
Chapter 9. Figure 5. Highly methylated DNA can be enriched greater than 40-fold.
Chapter 9. Figure 6. Melt-curves of amplified DNA targets.
Chapter 9. Figure 7. Amplification bias and minimization in a model system.
Chapter 9. Figure 8. Bisulfite converted DNA titration curve from 0% to 100% methylation.
Chapter 9. Figure 9. Bisulfite converted DNA titration curve from 0% to 100% methylation.
Chapter 9. Figure 10. Overview of MAGnify ChIP workflow vs. conventional ChIP protocols.
Chapter 9. Figure 11. 3 million Human 293Gt cells were fixed with 1% formaldehyde for 10 minutes at room temperature.
Chapter 9. Figure 12. Serial dilution of input DNA were amplified using SYBR GreenER PCR reagents.
Chapter 9. Figure 13. Results of typical ChIP experiment.
Chapter 9. Figure 14. Results of typical ChIP experiment.
Chapter 9. Figure 15. The NCode miRNA Universal qRT-PCR workflows.
Chapter 9. Figure 16. miRNA detection from total RNA or enriched miRNA starting materials.
Chapter 9. Figure 17. Illustration of Stem-loop Rt-qPCR method.
Chapter 9. Figure 18. Schematic of Megaplex Preamp TaqMan MicroRNA Assays for single cell gene expression profiling.
Chapter 9. Figure 19. Single cell miRNA and mRNA expression profile in 90 individual mES, mEB and somatic cells.

Chapter 10: High Resolution Melting Analysis

Chapter 10. Figure 1. Dual hybridization probe (HybProbe) technology for SNP genotyping.
Chapter 10. Figure 2. Basic software analysis for HRMA genotyping.
Chapter 10. Figure 3. Advanced software analysis for HRMA heterozygote scanning.
Chapter 10. Figure 4. Unlabeled probe chemistry.
Chapter 10. Figure 5. Snapback primer chemistry.
Chapter 10. Figure 6. The effect of secondary structure at the 3' end of a primer.
Chapter 10. Figure 7. The effect of varying template concentration and purity, observed by late amplification and poor reproducibility of melting curves.

Chapter 11: Microfluidic Emulsion PCR

Chapter 11. Figure 1. Emulsified PCR reactions generated by horizontal agitation of a stainless steel mixing ball.
Chapter 11. Figure 2. Picoliter-scale droplets on-chip prior to performing real-time reverse-transcription PCR.
Chapter 11. Figure 3. Distribution of genomic copies in droplets under a Poisson process for an average dilution of one copy per droplet.
Chapter 11. Figure 4. Distribution of genomic copies in droplets under a Poisson process for an average dilution of 0.5 copies per droplet.
Chapter 11. Figure 5. Observed percent amplification verses theory.
Chapter 11. Figure 6. Inverse relationship between fragment length and polony diameter amplified by bridge amplification at three acrylamide concentrations.
Chapter 11. Figure 7. Inverse relationship between fragment length and amplification yield on emulsified DNA Capture beads.
Chapter 11. Figure 8. Process flow from bulk solid-phase emPCR to next-gen sequencing.