1887

Chapter 4 : Real-Time PCR and Melting Analysis

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in
Zoomout

Real-Time PCR and Melting Analysis, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555816834/9781555814977_Chap04-1.gif /docserver/preview/fulltext/10.1128/9781555816834/9781555814977_Chap04-2.gif

Abstract:

This chapter talks about the fundamentals of real-time PCR and melting analysis. It draws an analogy between bacterial growth and PCR and then considers the kinetic requirements of PCR. It provides an overview of real-time instrumentation and fluorescent indicators. It considers methods for detection, quantification, and melting analysis, including high-resolution melting analysis. Real-time PCR with melting analysis can integrate the detection, quantification, and analysis of microbes in one rapid assay. In real-time PCR and melting analysis, fluorescence acquisition may extend the time required in instruments with high noise and/or low fluorescence sensitivity. All real-time PCR instruments monitor sample fluorescence during thermal cycling and are available from many manufacturers. A wide variety of different instruments, dyes, and probe designs are available for real-time PCR. The chapter discusses some of the methods commonly used for detection, quantification, and melting analysis. It also focuses on melting-curve analysis. Inspection of continuous plots during real-time PCR suggests that hybridization information can be extracted during temperature cycling when dyes or hybridization probes are used. Continued improvements in speed, integration of high-resolution melting analysis, and adoption of simple hybridization probe techniques like unlabeled probe and snapback primers will expand the reach of this powerful technique in the coming years.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4

Key Concept Ranking

Real-Time PCR
0.9113761
Quantitative PCR
0.76595765
Hybridization Techniques
0.6832644
Analytical Methods
0.6657128
0.9113761
Highlighted Text: Show | Hide
Loading full text...

Full text loading...

Figures

Image of FIGURE 1
FIGURE 1

Model exponential and logistic curves for bacterial growth and PCR. Doubling times of 20 min and 30 s are assumed for bacteria and PCR, respectively. That is, given the equation N = N , r is 0.0347 min for bacteria and 1.386 min for PCR. The carrying capacity for bacteria was set at 10/ml. Assuming that PCR is primer limited at one-third the primer concentration ( Table 1 ), a carrying capacity of 10 copies of PCR product/10 µl was used. The shapes of the curves for bacteria and DNA are identical, with only the axis scales specific to each method. Starting with a single bacterium, growth plateaus after 11 to 12 h, while PCR takes only 23 min (46 cycles) to amplify a single copy to saturation.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 2
FIGURE 2

Ideal logistic fit to real-time PCR data. The logistic equation = [ K/ + (K – )] was fit by nonlinear least-squares regression (Delta graph 4.0) to data from cycles 12 to 33 of a real-time PCR amplification. SYBR Green I was used to monitor the amplification of β-actin cDNA ( ). The best fit for the rate constant (γ) was 0.579 reciprocal cycle ( ), giving an apparent PCR efficiency of 0.78.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 3
FIGURE 3

Equilibrium and kinetic paradigms of PCR. Each paradigm focuses on three reactions (denaturation, annealing, and extension) during each PCR cycle. In the conventional equilibrium paradigm for PCR (left), each reaction occurs at a single temperature over a certain time period. Temperature transitions are not considered. In contrast, in the kinetic paradigm (right), the temperature is always changing. Each reaction occurs over a temperature range, rates depend on temperature, and more than one process can occur simultaneously. Reprinted from ( ) with permission of the publisher.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 4
FIGURE 4

Dynamic range of DNA dyes in real-time PCR. PCR was performed in the presence of optimal amounts of SYBR Green I (1:10,000 dilution), ethidium bromide (5 µg/ml), or acridine orange (3 µg/ml). A prototype real-time instrument ( ) was modified to detect each dye at the appropriate wavelength (SYBR Green I, 520 to 550 nm; ethidium bromide, 580 to 620 nm; acridine orange, 520 to 560 and 610 to 650 nm as a ratio). A 110-bp fragment of β-globin was amplified from 10 copies of a larger β-globin amplicon by 30 temperature cycles of 90°C for 0 s and 59°C for 20 s. Fluorescence was monitored every 0.2 s, but only data from cycles 1 and 30 are displayed. The relative fluorescence from each sample was normalized to 1.0 at the annealing temperature of cycle 1 (shown as a solid circle). At the beginning of amplification, the fluorescence is low and the cooling curve is identical to the heating curve. At cycle 30, fluorescence is increased at most temperatures, reflecting the greater amount of double-stranded product present. The cooling and heating curves trace different paths, and product melting is apparent as a sharp drop in fluorescence during heating.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 5
FIGURE 5

Heterozygote detection with different dyes. Heterozygote detections by three saturating dyes (LCGreen, SYTO 9, and EvaGreen) and the nonsaturating dye SYBR Green I are compared. (A) A short amplicon surrounding the F508del site of CFTR was amplified, using heterozygous F508del DNA as a template ( ). (B) High-resolution melting analysis (HR-1; Idaho Technology) was performed, and the data were plotted as a negative derivative after normalization and curve overlay. In each case, optimal dye concentrations that minimized PCR inhibition and maximized heteroduplex detection were used. The major peak on the right is composed of homoduplexes, while the smaller peak on the left (when present) consists of heteroduplexes. Although heteroduplexes were not observed with SYBR Green I, increasing heteroduplex signals were detected with EvaGreen, SYTO 9, and LCGreen. Reprinted from reference with permission of the publisher.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 6
FIGURE 6

Typical real-time PCR amplifications monitored with SYBR Green I, hydrolysis probes, and hybridization probes ( ). Both once-per-cycle and continuously monitored displays are shown. Note the hybridization information inherent in reactions monitored with SYBR Green I and hybridization probes. dsDNA, double-stranded DNA.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 7
FIGURE 7

Continuous monitoring of rapid-cycle PCR. A 110-bp β-globin fragment was amplified from human genomic DNA by cycling between 60°C and 90°C over 15 min in the presence of SYBR Green I. In the center is a three-dimensional plot of temperature, time, and fluorescence. On the bottom is the temperature profile over time. On the right is the fluorescence profile over time. At upper left is a fluorescence-versus-temperature plot that continuously monitors hybridization. Reprinted from ( ) with permission of the publisher.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 8
FIGURE 8

A typical rapid-cycle real-time PCR amplification. Forty cycles were completed in a little over 15 min with melting requiring an additional 4 to 5 min. The target was a 250-bp fragment of exon 2 of the PIGA gene amplified from human genomic DNA. Detection and quantification are enabled by monitoring fluorescence once each cycle at the end of extension (solid squares). Amplification is immediately followed by melting-curve acquisition by heating at 0.2°C/s. Melting-curve analysis identifies PCR products and sequence alterations by their melting temperatures. The original melting-curve data (solid line) can also be plotted as a derivative melting curve (dotted line). Reprinted from reference with permission from the American Society of Investigative Pathology and the Association for Molecular Pathology.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 9
FIGURE 9

Different real-time PCR curve shapes. The curves in panels A and B are drift artifacts from negative samples that have been magnified for easy inspection. Curves C to F are all from positive samples.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 10
FIGURE 10

Automatic identification of positive and negative samples by dynamic baseline selection and a confidence band. The baselines (dotted lines) are extrapolated from the data segment with slope closest to zero. The confidence bandwidth is determined by the variance of points within the data segment. Drifts from negative samples (A) are distinguished from positive samples (B).

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 11
FIGURE 11

A typical amplification curve (F) along with its first (F′) and second (F′) derivatives. The curve shape is partly characterized by the sequential ordering of the minimum value, the second-derivative maximum, the firstderivative maximum, the second-derivative minimum, and the maximum value.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 12
FIGURE 12

Real-time PCR monitored by hydrolysis probe fluorescence each cycle ( ). The average number of initial template copies varied from 0.15 to 15,000. Samples were run in duplicate. The thin lines indicate the log-linear portion of each curve. The fluorescence obtained from one of the samples averaging 0.15 copy suggests that tubes with one copy of target can be distinguished from tubes with no copies.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 13
FIGURE 13

SYBR Green I derivative melting curves of 24 samples containing an average of one template copy ( ). The positive samples are clearly distinguished from the negative samples by the presence of a peak corresponding to the expected product .

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 14
FIGURE 14

The probability of 0 copies being present, given an average number of copies per tube. Data were extracted from Poisson distribution tables and are presented on both linear and log scales.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 15
FIGURE 15

Absolute real-time PCR quantification. (A) The logistic equation was used to generate idealized titration curves with a PCR efficiency of 1.0. The logistic limit or carrying capacity was progressively decreased at 10 copies and below to simulate actual observation ( ). (B) The fractional cycle number of each curve where the fluorescence rises is plotted against the log of the initial template concentration. Since the initial template concentration is the independent variable, the and axes are usually reversed from those shown.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 16
FIGURE 16

Relative quantification by real-time PCR. In panel A, the relative copy number of a PCR target in an experimental sample and a control sample is determined by real-time PCR. The fluorescence in the sample with more copies is the first to rise above the baseline. The second-derivative maximum (Cq) of each curve is shown as a vertical dotted line. If the PCR efficiency of the target is E, then the copy number of the experimental sample relative to the control sample is E + 1 raised to the difference between Cq values (ΔC = C –C). This calculation assumes that the PCR efficiency and the starting amount of material (DNA or cDNA) in each sample is the same. As the efficiency approaches 1.0, the relative copy number approaches 2. In panel B, the relative copy number of the test target (Test) is normalized to a reference target (Ref). Any difference in the amount of starting material is normalized by the results of the reference target. This method assumes that the reference target is invariant between samples and that the PCR efficiency for each target does not vary between samples. As the efficiency of both targets approaches 1.0, the relative copy number of the test target with reference gene normalization approaches 2 raised to the ΔC –ΔC power.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 17
FIGURE 17

Calculation of the second-derivative maximum. The second derivative of the fluorescence-versus-cycle plot is first obtained by Savitzky-Golay polynomial estimation. Then, a quadratic is fit around the maximum cycle (five points) to obtain the second-derivative maximum as a fractional cycle number.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 18
FIGURE 18

The precision of different fractional cycle number (Cq) methods. Thirty-two identical amplifications from human genomic DNA were analyzed by five different methods to estimate Cq precision (LightCycler 1.5; Roche Applied Science). Histograms reveal outlying samples in all methods except the second-derivative method. The second-derivative method depends on the curve shape, not on the level of fluorescence. Cq standard deviations from this data are shown in Table 3 .

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 19
FIGURE 19

Fluorescent DNA melting analysis. In panel A, the original fluorescence data show a linear decrease of fluorescence at low temperatures, followed by a rapid decrease centered around the . Fluorescence is low at high temperature when the DNA is single stranded. In panel B, the original data are normalized between 0 and 100% after background subtraction so that the data are horizontal outside the transition.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 20
FIGURE 20

multiplexing for species identification ( ). After PCR and melting, the derivative melting peaks correlate with (a), (b), (c), (d), and an internal control (e). GC-rich tails were added to the 5′ end of primers for and to increase their s. Reprinted from ( ) with permission of the publisher.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 21
FIGURE 21

High-resolution genotyping of a single-nucleotide variant within a 544-bp PCR product amplified from human genomic DNA ( ). Duplicate samples of each genotype (CC, TC, and TT) are shown. The data were normalized, and temperature was overlaid to superimpose the curves between 10% and 20% fluorescence. Two melting domains were present. Melting curves for the different genotypes were similar in the higher melting temperature domain but differed in the lower melting domain. The inset magnifies the boxed area. Reprinted from ( ) with permission of the publisher.

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 22
FIGURE 22

Variant typing by melting analysis. Primer and probe designs are shown on the left with typical data on the right. Dual (A) and single (B) hybridization probes use covalent fluorescent labels (asterisks), and typing is solely derived from the probe signal. Unlabeled probes (C) and snapback primers (D) require no covalent labels because fluorescence is provided by the saturation dye, LCGreen. Both probe and PCR product melting transitions are observed and can contribute to typing. Any free 3′ ends on the probes are terminated with a phosphate (Pi) or other blocker to prevent probe extension by the polymerase. The snapback primer (D) incorporates an unlabeled probe into the 5′ end of one primer, generating a self-probing amplicon that forms a hairpin. In panel E, no probe is present, but typing of the PCR product is still possible by high-resolution melting. High-resolution melting identifies heterozygotes by a change in curve shape and distinguishes homozygotes by .

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Permissions and Reprints Request Permissions
Download as Powerpoint

References

/content/book/10.1128/9781555816834.ch04
1. Bernard, P. S.,, R. S. Ajioka,, J. P. Kushner, and, C. T. Wittwer. 1998. Homogeneous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes. Am. J. Pathol. 153:10551061.
2. Bustin, S.,, J. Garson,, J. Hellemans,, J. Huggett,, M. Kubista,, R. Mueller,, T. Nolan,, M. Pfaffl,, G. Shipley,, J. Vandesompele, and, C. Wittwer. 2009. The MIQE guidelines: minimal information for publication of quantitative real-time PCR experiments. Clin. Chem. 55:611622.
3. Cheng, J. C.,, C. L. Huang,, C. C. Lin,, C. C. Chen,, Y. C. Chang,, S. S. Chang, and, C. P. Tseng. 2006. Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR. Clin. Chem. 52:19972004.
4. Crockett, A. O.,, and C. T. Wittwer. 2001. Fluorescein-labeled oligonucleotides for real-time PCR: using the inherent quenching of deoxyguanosine nucleotides. Anal. Biochem. 290:8997.
5. Dames, S.,, R. L. Margraf,, D. C. Pattison,, C. T. Wittwer, and, K. V. Voelkerding. 2007. Characterization of aberrant melting peaks in unlabeled probe assays. J. Mol. Diagn. 9:290296.
6. Dames, S.,, D. C. Pattison,, L. K. Bromley,, C. T. Wittwer, and, K. V. Voelkerding. 2007. Unlabeled probes for the detection and typing of herpes simplex virus. Clin. Chem. 53:18471854.
7. Dujols, V. E.,, N. Kusukawa,, J. T. McKinney,, S. F. Dobrowolski, and, C. T. Wittwer. 2006. High-resolution melting analysis for scanning and genotyping, p. 157-171. In M. T. Dorak (ed.), Real-Time PCR. Garland Science, New York, NY.
8. Erali, M.,, R. Palais, and, C. T. Wittwer. 2008. SNP genotyping by unlabeled probe melting analysis, p. 199-206. In O. Seitz and, A. Marx (ed.), Molecular Beacons: Signalling Nucleic Acid Probes, Methods and Protocols, vol. 429. Humana Press, Totowa, NJ.
9. Erali, M.,, J. I. Pounder,, G. L. Woods,, C. A. Petti, and, C. T. Wittwer. 2006. Multiplex single-color PCR with amplicon melting analysis for identification of Aspergillus species. Clin. Chem. 52:14431445.
10. Erali, M.,, K. V. Voelkerding, and, C. T. Wittwer. 2008. High resolution melting applications for clinical laboratory medicine. Exp. Mol. Pathol. 85:5058.
11. Espy, M. J.,, J. R. Uhl,, L. M. Sloan,, S. P. Buckwalter,, M. F. Jones,, E. A. Vetter,, J. D. Yao,, N. L. Wengenack,, J. E. Rosenblatt,, F. R. Cockerill III, and, T. F. Smith. 2006. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin. Microbiol. Rev. 19:165256.
12. Farrar, J. S.,, G. H. Reed, and, C. T. Wittwer. 2009. High resolution melting curve analysis for molecular diagnostics, p. 229-245. In G. P. Patrinos and, W. Ansorge (ed.), Molecular Diagnostics, 2nd ed. Elsevier, Burlington, MA.
13. Ghadessy, F. J.,, J. L. Ong, and, P. Holliger. 2001. Directed evolution of polymerase function by compartmentalized self-replication. Proc. Natl. Acad. Sci. USA 98:45524557.
14. Gudnason, H.,, M. Dufva,, D. D. Bang, and, A. Wolff. 2007. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Res. 35:e127.
15. Gundry, C. N.,, S. F. Dobrowolski,, Y. R. Martin,, T. C. Robbins,, L. M. Nay,, N. Boyd,, T. Coyne,, M. D. Wall,, C. T. Wittwer, and, D. H. Teng. 2008. Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons. Nucleic Acids Res. 36:34013408.
16. Gundry, C. N.,, J. G. Vandersteen,, G. H. Reed,, R. J. Pryor,, J. Chen, and, C. T. Wittwer. 2003. Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes. Clin. Chem. 49:396406.
17. Herrmann, M. G.,, J. D. Durtschi,, L. K. Bromley,, C. T. Wittwer, and, K. V. Voelkerding. 2006. Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. Clin. Chem. 52:494503.
18. Herrmann, M. G.,, J. D. Durtschi,, L. K. Bromley,, C. T. Wittwer, and, K. V. Voelkerding. 2007. Instrument comparison for heterozygote scanning of single and double heterozygotes: a correction and extension of Herrmann et al. Clin. Chem. 52:494503. (Author’s correction, 53:150–152.)
19. Herrmann, M. G.,, J. D. Durtschi,, C. T. Wittwer, and, K. V. Voelkerding. 2007. Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping. Clin. Chem. 53:15441548.
20. Higuchi, R.,, G. Dollinger,, P. S. Walsh, and, R. Griffith. 1992. Simultaneous amplification and detection of specific DNA sequences. Bio/Technology 10:413417.
21. Jepsen, J. S.,, M. D. Sorensen, and, J. Wengel. 2004. Locked nucleic acid: a potent nucleic acid analog in therapeutics and biotechnology. Oligonucleotides 14:130146.
22. Kutyavin, I. V.,, I. A. Afonina,, A. Mills,, V. V. Gorn,, E. A. Lukhtanov,, E. S. Belousov,, M. J. Singer,, D. K. Walburger,, S. G. Lokhov,, A. A. Gall,, R. Dempcy,, M. W. Reed,, R. B. Meyer, and, J. Hedgpeth. 2000. 3′-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 28:655661.
23. Lay, M. J.,, and C. T. Wittwer. 1997. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin. Chem. 43:22622267.
24. Li, H.,, G. Xue, and, E. S. Yeung. 2001. Selective detection of individual DNA molecules by capillary polymerase chain reaction. Anal. Chem. 73:15371543.
25. Li, J.,, F. Wang,, H. Mamon,, M. H. Kulke,, L. Harris,, E. Maher,, L. Wang, and, G. M. Makrigiorgos. 2006. Antiprimer quenching-based real-time PCR and its application to the analysis of clinical cancer samples. Clin. Chem. 52:624633.
26. Li, Q.,, G. Luan,, Q. Guo, and, J. Liang. 2002. A new class of homogeneous nucleic acid probes based on specific displacement hybridization. Nucleic Acids Res. 30:E5.
27. Lin, J. H.,, C. P. Tseng,, Y. J. Chen,, C. Y. Lin,, S. S. Chang,, H. S. Wu, and, J. C. Cheng. 2008. Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis. J. Clin. Microbiol. 46:10901097.
28. Livak, K. J.,, S. J. Flood,, J. Marmaro,, W. Giusti, and, K. Deetz. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 4:357362.
29. Lun, M. F.,, R. W. K. Chiu,, K. C. A. Chan,, T. Y. Leung,, T. K. Lau, and, Y. M. D. Lo. 2008. Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin. Chem. 54:16641672.
30. Lyon, E.,, and C. T. Wittwer. 2009. LightCycler technology in molecular diagnostics. J. Mol. Diagn. 11:93101.
31. Margraf, R. L.,, R. Mao, and, C. T. Wittwer. 2006. Masking selected sequence variation by incorporating mismatches into melting analysis probes. Hum. Mutat. 27:269278.
32. Morrison, T.,, J. Hurley,, J. Garcia,, K. Yoder,, A. Katz,, D. Roberts,, J. Cho,, T. Kanigan,, S. E. Ilyin,, D. Horowitz,, J. M. Dixon, and, C. J. Brenan. 2006. Nanoliter high throughput quantitative PCR. Nucleic Acids Res. 34:e123.
33. Morrison, T. B.,, J. J. Weis, and, C. T. Wittwer. 1998. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Bio-Techniques 24:954958, 960, 962.
34. Nazarenko, I. A.,, S. K. Bhatnagar, and, R. J. Hohman. 1997. A closed tube format for amplification and detection of DNA based on energy transfer. Nucleic Acids Res. 25:25162521.
35. Reed, G. H.,, J. O. Kent, and, C. T. Wittwer. 2007. High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 8:597608.
36. Ririe, K. M.,, R. P. Rasmussen, and, C. T. Wittwer. 1997. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245:154160.
37. Rohner, P.,, B. Pepey, and, R. Auckenthaler. 1996. Comparative evaluation of BACTEC aerobic Plus/F and SeptiChek Release blood culture media. J. Clin. Microbiol. 34:126129.
38. Schmittgen, T. D.,, and K. J. Livak. 2008. Analyzing realtime PCR data by the comparative C(T) method. Nat. Protoc. 3:11011108.
39. Seipp, M. T.,, J. D. Durtschi,, M. A. Liew,, J. Williams,, K. Damjanovich,, G. Pont-Kingdon,, E. Lyon,, K. V. Voelkerding, and, C. T. Wittwer. 2007. Unlabeled oligonucleotides as internal temperature controls for genotyping by amplicon melting. J. Mol. Diagn. 9:284289.
40. Seipp, M. T.,, D. Pattison,, J. D. Durtschi,, M. Jama,, K. V. Voelkerding, and, C. T. Wittwer. 2008. Quadruplex genotyping of F5, F2, and MTHFR variants in a single closed tube by high-resolution amplicon melting. Clin. Chem. 54:108115.
41. Selvapandiyan, A.,, K. Stabler,, N. A. Ansari,, S. Kerby,, J. Riemenschneider,, P. Salotra,, R. Duncan, and, H. L. Nakhasi. 2005. A novel semiquantitative fluorescence-based multiplex polymerase chain reaction assay for rapid simultaneous detection of bacterial and parasitic pathogens from blood. J. Mol. Diagn. 7:268275.
42. Spurgeon, S. L.,, R. C. Jones, and, R. Ramakrishnan. 2008. High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PLoS ONE 3:e1662.
43. Stephens, A. J.,, J. Inman-Bamber,, P. M. Giffard, and, F. Huygens. 2008. High-resolution melting analysis of the spa repeat region of Staphylococcus aureus. Clin. Chem. 54:432436.
44. Svanvik, N.,, G. Westman,, D. Wang, and, M. Kubista. 2000. Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution. Anal. Biochem. 281:2635.
45. Tan, S. S.,, and J. H. Weis. 1992. Development of a sensitive reverse transcriptase PCR assay, RT-RPCR, utilizing rapid cycle times. PCR Methods Appl. 2:137143.
46. Todd, A. V.,, C. J. Fuery,, H. L. Impey,, T. L. Applegate, and, M. A. Haughton. 2000. DzyNA-PCR: use of DNAzymes to detect and quantify nucleic acid sequences in a real-time fluorescent format. Clin. Chem. 46:625630.
47. Tyagi, S.,, and F. R. Kramer. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14:303308.
48. Vogelstein, B.,, and K. W. Kinzler. 1999. Digital PCR. Proc. Natl. Acad. Sci. USA 96:92369241.
49. von Ahsen, N.,, C. T. Wittwer, and, E. Schutz. 2001. Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg(2+), deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas. Clin. Chem. 47:19561961.
50. Whitcombe, D.,, J. Theaker,, S. P. Guy,, T. Brown, and, S. Little. 1999. Detection of PCR products using self-probing amplicons and fluorescence. Nat. Biotechnol. 17:804807.
51. Wittwer, C. T.,, and D. J. Garling. 1991. Rapid cycle DNA amplification: time and temperature optimization. BioTechniques 10:7683.
52. Wittwer, C. T.,, and M. G. Herrmann. 1999. Rapid thermal cycling and PCR kinetics, p. 211-229. In M. Innis,, D. Gelfand, and, J. Sninsky (ed.), PCR Methods Manual. Academic Press, San Diego, CA.
53. Wittwer, C. T.,, M. G. Herrmann,, C. N. Gundry, and, K. S. Elenitoba-Johnson. 2001. Real-time multiplex PCR assays. Methods 25:430442.
54. Wittwer, C. T.,, M. G. Herrmann,, A. A. Moss, and, R. P. Rasmussen. 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques 22:130131, 134–138.
55. Wittwer, C. T.,, and N. Kusukawa. 2004. Real-time PCR, p. 71-84. In D. H. Persing,, F. C. Tenover,, J. Versalovic,, Y. W. Tang,, E. R. Unger,, D. A. Relman, and, T. J. White (ed.), Diagnostic Molecular Microbiology: Principles and Applications. ASM Press, Washington, DC.
56. Wittwer, C. T.,, R. P. Rasmussen, and, K. M. Ririe. 2010. Rapid PCR and melting analysis, p. 48-69. In S. A. Bustin (ed.), The PCR Revolution: Basic Technologies and Applications. Cambridge University Press, New York, NY.
57. Wittwer, C. T.,, G. B. Reed, and, K. M. Ririe. 1994. Rapid cycle DNA amplification, p. 174-181. In I. K. Mullis,, F. Ferre, and, R. Gibbs (ed.), The Polymerase Chain Reaction. Springer-Verlag, Deerfield Beach, FL.
58. Wittwer, C. T.,, G. H. Reed,, C. N. Gundry,, J. G. Vandersteen, and, R. J. Pryor. 2003. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin. Chem. 49:853860.
59. Wittwer, C. T.,, K. M. Ririe,, R. V. Andrew,, D. A. David,, R. A. Gundry, and, U. J. Balis. 1997. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. BioTechniques 22:176181.
60. Wittwer, C. T.,, K. M. Ririe, and, R. P. Rasmussen. 1998. Fluorescence monitoring of rapid cycle PCR for quantification, p. 129-144. In F. Ferre (ed.), Gene Quantification. Birkhauser, New York, NY.
61. Zhou, L.,, R. J. Errigo,, H. Lu,, M. A. Poritz,, M. T. Seipp, and, C. T. Wittwer. 2008. Snapback primer genotyping with saturating DNA dye and melting analysis. Clin. Chem. 54:16481656.
62. Zhou, L.,, A. N. Myers,, J. G. Vandersteen,, L. Wang, and, C. T. Wittwer. 2004. Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye. Clin. Chem. 50:13281335.
63. Zhou, L.,, L. Wang,, R. Palais,, R. Pryor, and, C. T. Wittwer. 2005. High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clin. Chem. 51:17701777.
64. Zipper, H.,, H. Brunner,, J. Bernhagen, and, F. Vitzthum. 2004. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Res. 32:e103.

Tables

Generic image for table
TABLE 1

Typical reactant amounts in PCR (10-µl reaction mixture)

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Generic image for table
TABLE 2

Correlation between PCR efficiency and amplification curve spacing

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4
Generic image for table
TABLE 3

Real-time PCR precision

Citation: Wittwer C, Kusukawa N. 2011. Real-Time PCR and Melting Analysis, p 63-82. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch4

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error