• View in gallery

    Multivariate regression analysis of Δ Ct for short vs long amplicons from the same nine double-stranded DNA templates based on three variables. 1) Absolute difference in basepairs between short and long amplicons from the same double-stranded DNA template (Δ length). 2) Relative difference in guanine/cytosine (GC) content between short and long amplicons (Δ GC%). 3) Absolute differences in adenine/thymine (AT) and GC content (in basepairs) for the long amplicon minus the same differences for the corresponding short amplicon (the AT-GC differential is shown in Supplemental Table 3). Among these variables, the AT-GC differential best explained the change in Cts (Table in Supplemental Figure 1). Analysis based on the AT-GC differential showed that this parameter alone accounted for 90% of the change in Ct (P < 0.001). The amplicons in this analysis were generated using the primers listed in Supplemental Table 2.

  • View in gallery

    Real-time polymerase chain reaction using SYBR Green and comparing adenine/thymine (AT)–rich (left set of curves) and AT-poor (right set of curves) double-stranded DNA synthetic templates of 150 bp. This figure appears in color at www.ajtmh.org.

  • View in gallery

    Real-time polymerase chain reaction using SYBR Green and comparing a 76-bp double-stranded DNA template containing 50% adenine/thymine (AT) (right set of curves) with a 150-bp double-stranded template containing 50% AT (left set of curves). This figure appears in color at www.ajtmh.org.

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Estimation of Copy Number using SYBR Green: Confounding by AT-rich DNA and by Variation in Amplicon Length

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  • 1 Departments of Tropical Medicine and Medicine, and the Center for Infectious Diseases, Tulane University Health Sciences Center, New Orleans, Louisiana; Department of Biology, Faculty of Science and Technology, University of Bamako, Bamako, Mali

Although SYBR Green is used to estimate copy number, its fluorescence varies with amplicon length and adenine/thymine (AT) content. As a result, threshold cycle (Ct) values obtained using real-time polymerase chain reaction (PCR) are lower for longer amplicons (P < 0.001) and amplicons with greater AT content (P < 0.001). In contrast, neither amplicon length nor AT content affects the Ct with TaqMan probes or LUX-labeled primers. Because SYBR Green yields lower Cts with AT-rich templates and longer templates, it overestimates copy number for those templates. Therefore, sequence-specific methods such as TaqMan probes or LUX-labeled primers should be considered when using real-time PCR to estimate copy number if the amplicons generated are AT-rich or vary in length.

INTRODUCTION

To estimate copy number, a real-time polymerase chain reaction (PCR) uses one of two fluorescence-based detection methods: fluorophore-labeled oligonucleotide probes or fluorescent dyes. Oligonucleotide probes (TaqMan probes, LUX-labeled primers) bind specifically to the sequences amplified during the PCR; they are valuable because of their specificity and have fluorophores attached to them that permit their detection. However, oligonucleotide probes can be difficult to design, are more expensive, and frequently require optimization. In contrast, fluorescent dyes that intercalate and bind in the minor groove such as SYBR Green bind to all amplicons, are more economical, obviate the need for additional primer/ probe design, and fluoresce effectively with double-stranded DNA (dsDNA).1 Because of its low cost, compatibility with melt curve analyses, and ease of use, SYBR Green is used frequently for real-time PCR.2 On the basis of a survey performed recently by Bio-Rad (Hercules, CA), SYBR Green is used for ≥ 85% of the real-time PCRs performed to estimate copy number (Lawson M, unpublished data).

Despite the popularity of SYBR Green, unresolved questions about its use include the effects of amplicon length and nucleic acid composition on the threshold cycle (Ct). Recent reports suggest that SYBR Green may have greater affinity for adenine/thymine (AT)–rich DNA than guanine/cytosine (GC)–rich DNA,35 and that its binding may be greater with longer amplicons.2 We used Plasmodium falciparum DNA and synthetic dsDNA templates to perform the studies reported here, which compared Cts obtained with SYBR Green with those obtained with sequence-specific strategies (Taq-Man probes and LUX-labeled primers) for amplicons that varied in length and AT content.

MATERIALS AND METHODS

DNA sources and preparations.

Control P. falciparum template DNA was obtained from cloned, cultured parasites6 using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA). Isolates cloned by limiting dilution and used as controls for the polymorphic merozoite surface protein 1 (msp1) Block 2 allotypes of P. falciparum were Indochina I/CDC for MAD20 and Haiti 135 for K1.7,8 Genomic DNA was extracted from adult female Anopheles gambiae G3 mosquitoes (MRA-112, MR4; American Type Culture Collection, Manassas, VA) with the QIAamp DNeasy Tissue Kit (Qiagen) by using the insect protocol recommended by the manufacturer. Control human genomic DNA was isolated from buccal swabs obtained from volunteers (JMC, BDB) using the QIAamp DNA Mini Kit (Qiagen). DNA template concentrations were estimated from a standard curve of fluorescence versus DNA concentration, using γ DNA (51% AT)9 in a series of 10-fold dilutions from 1,000 ng/mL to 1 ng/mL with PicoGreen dye (Molecular Probes, Eugene, OR) and a VersaFluor 170-2402 fluorometer (Bio-Rad). Synthetic ds-DNA templates (Supplemental Table 1, available at www.ajtmh.org) were designed manually and synthesized commercially (Midland Certified Reagent Company, Midland, TX). Because of the potentially biased binding of single-strand nucleic acid reagents (e.g., OliGreen; Invitrogen, Carlsbad, CA),10 synthetic templates were suspended at 100 nM concentrations in RNase-free water; those concentrations were then verified with sequence-specific strategies (LUX primers or TaqMan probes) by using real-time PCR.

Primers and probes for real-time PCR (Supplemental Table 2, available at www.ajtmh.org) were designed using the Primer311 or Beacon Designer software, version 2.03 (Premier Biosoft International, Palo Alto, CA) (available at http://www.premierbiosoft.com/molecular_beacons/index.html) with manual editing as needed. Unlabeled primers and fluorophore-labeled probes were obtained from Integrated DNA Technologies (Coraville, IA), and LUX-labeled primers were obtained from Invitrogen. When sequences were available from GenBank, the database of the National Center for Biotechnology Information (Bethesda, MD)1215 was used to design primers and probes. Partial sequences for the Block 2 region of the P. falciparum msp1 gene (K1, MAD20, and hybrid MAD20/RO33 clones) were obtained from direct sequencing of PCR products by the Sequencing Core at the Tulane University Center for Gene Therapy using primers described elsewhere,16,17 and have been submitted to GenBank (accession nos. DQ404192, DQ404191, and DQ447647).

Real-time PCR amplification was performed with the Bio-Rad iCycler using the 2X iQ supermix or the 2X iQ SYBR Green supermix (Bio-Rad). Template DNA was added to 50 μL PCR mixtures in aliquots of 3–5 μL for extracted genomic DNA and 1 μL for synthetic template DNA. The PCR mixtures were then subjected to initial denaturation at 95°C for 3 minutes, followed by 45 cycles of denaturation at 94°C for 30 seconds, annealing for 60 seconds, and extension at 72°C for 60 seconds (annealing temperatures were optimized for each primer pair [range = 53–59°C] to match the efficiencies of the experimental templates and primers). Fluorescence measurements were obtained during the annealing step with TaqMan probes and during the elongation step with LUX primers and SYBR Green. Each template-primer pair was tested in replicates of at least nine to estimate the mean Ct. Single amplicons of the expected size were obtained with matched template-primer probe sets; no amplicons were obtained with unmatched template-primer probe sets or in the absence of template DNA.

Sequence and statistical analysis.

DNA sequences were analyzed with the EditSeq program (Lasergene software; DNASTAR, Madison, WI) or Discovery Studio Gene version 1.5 (Accelrys, San Diego, CA), and mean Cts were compared using the t-test. Multivariate regression analysis was performed using the generalized linear model, and statistical analyses were performed with SPSS software version 12.0 (SPSS, Chicago, IL).

RESULTS

Standard curves and efficiency of replication.

Standard curves (regression lines) plotted the log10 dilution of copy number versus Ct. Linear correlation coefficients (r2) were between 0.990 and 0.999 for all templates tested. Estimates of efficiency (the degree to which replication increased the number of amplicons by the expected two-fold increment [100% efficiency] in each cycle),18 which indicated that the efficiencies of these assays ranged from 80% to 100%. Efficiencies for matched primer pairs were within ≤ 5%.

Effect of amplicon length on Cts with SYBR Green.

Primers were designed to yield short and long amplicons from Block 2 of msp1 with two P. falciparum parasites (one K1 clone, one MAD20 clone, Table 1). For K1, a single reverse primer (K1R) was used with two forward primers (K1F-Short, K1F-Long) to produce short and long amplicons (149 and 221 bp). Likewise, for MAD20, a single reverse primer (MAD20R) was used with two forward primers (MAD20F-Short, MAD20F-Long) to produce short and long amplicons (174 and 226 bp). SYBR Green yielded lower Cts for long than short amplicons with K1 and MAD20 (24.29 versus 25.26 and 20.63 versus 21.35 cycles, respectively; P < 0.001; Table 1).

Effect of amplicon length on Cts with sequence-specific probes.

In these studies, the same primer sequences were used with an internal TaqMan probe for K1 and a LUX-labeled reverse primer for MAD20. Using the TaqMan probe, we observed that there were no differences in Cts between long and short K1 amplicons (26.29 versus 25.88 cycles; P > 0.5, Table 2). Likewise, using a LUX-labeled reverse primer, we observed that there were no differences between long and short MAD20 amplicons (27.36 versus 27.28 cycles; P > 0.5, Table 2).

Effect of amplicon length and AT content on Ct.

The longer amplicons from P. falciparum DNA that yielded lower Cts with SYBR Green (Table 1) had greater absolute and relative AT content (Supplemental Table 3, available at www.ajtmh.org). In contrast, when dsDNA from other sources was examined, lower Cts were obtained for longer amplicons with only one of five genes: the human gene for tumor necrosis factor. The longer amplicons that produced lower Cts with SYBR Green had greater AT content than the shorter amplicons from the same templates. In contrast, the longer amplicons that did not produce lower Cts, had similar or lower AT content than the shorter amplicons from those templates (Supplemental Table 3).

Because differences in AT content could confound inter-pretation of the effects of amplicon size on Ct, we performed multivariate regression to evaluate the contributions of three variables: 1) absolute differences in basepairs between short and long amplicons from the same dsDNA templates (Δ length), 2) relative differences in GC content between short and long amplicons (Δ % GC), and 3) absolute differences in AT and GC content (in basepairs) for the long amplicon minus the same differences for the corresponding short amplicon (the AT-GC differential; Supplemental Table 3). The results of this analysis suggest that the AT-GC differential explains most of the differences in Ct (P = 0.025; Figure 1). When this analysis was repeated using only the AT-GC differential, it alone accounted for 90% of the variability in the Ct (r2 = 0.902, P < 0.001; Figure 1).

Effect of AT content and amplicon length of synthetic templates on Ct

To distinguish the effects of AT content and amplicon length, we designed synthetic dsDNA templates that were AT-rich and AT-poor (Table 3) and templates matched for relative and absolute AT content (Table 4). The lower Cts obtained for AT-rich templates with SYBR Green led to overestimates of copy number with 150 bp (Figure 2) and 450-bp templates (P < 0.001 for both comparisons), but not with 76-bp templates (Table 3). Likewise, longer dsDNA templates with the same relative AT concentration (50%) yielded lower Cts (Figure 3 and Table 4).

In contrast, real-time PCR with LUX-labeled primers showed no differences in Ct between AT-rich and AT-poor amplicons, or between amplicons with different numbers of AT basepairs, whether they were similar or different in length (Tables 3 and 4). The LUX-labeled primers also yielded similar Cts for amplicons of different lengths with the same number of AT basepairs (Table 4). Conversely, SYBR Green yielded different Cts for different size amplicons, even when those amplicons were matched for AT content (P < 0.001; Table 4).

DISCUSSION

Our initial observation (that copy number was overestimated with longer amplicons from P. falciparum DNA) suggested that longer amplicons might incorporate enough additional SYBR Green to decrease Ct and thus overestimate copy number. However, the AT-rich nature of P. falciparum DNA made it impossible to distinguish between the effects of AT content and amplicon length. Therefore, we prepared synthetic dsDNA templates and performed multivariate regression to identify the most important variables affecting Ct.

The multivariate regression analysis indicated that AT content (the AT-GC differential) was the most important deter-minant of Δ Ct (Figure 1). The studies of synthetic dsDNA templates with SYBR Green demonstrated that AT-rich templates yielded lower Cts than AT-poor templates (Figures 2 and 3) and resulted in estimates of copy number that differed by ≥ 4-fold (differences ≥ 2.0 in Ct, Tables 3 and 4). These results suggest that increased amplicon length affects Ct primarily because it is associated with increased numbers of AT basepairs. Although increased numbers of GC basepairs also reduce the Ct, the effect of additional GC basepairs is less than that of additional AT basepairs (Table 4).

The overestimation of copy number (from underestimation of the Ct) with AT-rich templates (Figures 2 and 3 and Tables 3 and 4) is consistent with a report by Zipper and others, which noted that SYBR Green produced twice as much fluorescence with dA-dT as with dG-dC homopolymers,3 but not with reports that have suggested greater binding to GC-rich DNA.19,20 Working from the perspective of Zipper and others,3 we have developed a model of this effect on the basis of the assumption that one AT basepair produces two units of fluorescence (estimated fluorescence units) for every unit of fluorescence produced by a GC basepair. The expected magnitudes of these differences were then plotted against experimentally determined Cts (Supplemental Figure 1, available at www.ajtmh.org). The apparent linear relationship between these variables (r2 = 0.7554), and the fact that there was no difference in Ct between AT-rich and AT-poor templates of 76 bp suggest that there is a threshold below which differences in AT-content do not affect Ct, and thus do not affect estimates of copy number or starting quantity.

These results indicate that SYBR Green overestimates copy number for amplicons with greater AT content or length. The effects of AT content apply not only to P. falciparum, but also to other AT-rich organisms such as Dictyostelium discoideum, vaccinia virus, Lactobacillus johnsonii, Bartonella species, and Mycoplasma mycoides.2125 The effects of amplicon length are broadly relevant to studies of humans, arthropod vectors, and microbial pathogens. The magnitude of these effects (Tables 3 and 4) is similar to (and can exceed) the ≥ 1.5-fold or 2.0-fold change criteria used to identify changes in genes expression with microarrays and real-time PCR (low density arrays).26

The results reported demonstrate that real-time PCR with SYBR Green can yield lower Cts, which lead to overestimation of copy number. These results suggest that AT content and amplicon length should be similar when performing real-time PCR to estimate copy number with SYBR Green. This confounding may be obviated by the use of sequence-specific strategies such as TaqMan probes or LUX-labeled primers. It can also be controlled by experiments in which the same amplicon serves as the reference and the experimental target.27

Table 1

Amplicons of different length that yield different Cts with SYBR Green*

Table 1
Table 2

Amplicons of different length that have similar Cts with sequence-specific probes and primers*

Table 2
Table 3

Amplicons from double-stranded DNA synthetic templates that vary in AT content and length*

Table 3
Table 4

Amplicons from synthetic double-stranded DNA templates of varying length matched for relative (%) and absolute (basepairs) AT content*

Table 4
Figure 1.
Figure 1.

Multivariate regression analysis of Δ Ct for short vs long amplicons from the same nine double-stranded DNA templates based on three variables. 1) Absolute difference in basepairs between short and long amplicons from the same double-stranded DNA template (Δ length). 2) Relative difference in guanine/cytosine (GC) content between short and long amplicons (Δ GC%). 3) Absolute differences in adenine/thymine (AT) and GC content (in basepairs) for the long amplicon minus the same differences for the corresponding short amplicon (the AT-GC differential is shown in Supplemental Table 3). Among these variables, the AT-GC differential best explained the change in Cts (Table in Supplemental Figure 1). Analysis based on the AT-GC differential showed that this parameter alone accounted for 90% of the change in Ct (P < 0.001). The amplicons in this analysis were generated using the primers listed in Supplemental Table 2.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 6; 10.4269/ajtmh.2008.79.887

Figure 2.
Figure 2.

Real-time polymerase chain reaction using SYBR Green and comparing adenine/thymine (AT)–rich (left set of curves) and AT-poor (right set of curves) double-stranded DNA synthetic templates of 150 bp. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 6; 10.4269/ajtmh.2008.79.887

Figure 3.
Figure 3.

Real-time polymerase chain reaction using SYBR Green and comparing a 76-bp double-stranded DNA template containing 50% adenine/thymine (AT) (right set of curves) with a 150-bp double-stranded template containing 50% AT (left set of curves). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 6; 10.4269/ajtmh.2008.79.887

*

Address correspondence to Donald J. Krogstad, Departments of Tropical Medicine and Medicine, and the Center for Infectious Diseases, Tulane University Health Sciences Center, 1430 Tulane Avenue, SL 17, J. Bennett Johnston Building, Room 510, New Orleans, LA 70112. E-mail: krogstad@tulane.edu

Note: Supplemental Table 1 (Sequences of Synthetic DNA Templates), Supplemental Table 2 (Primers and probes used for real-time PCR), Supplemental Table 3 (Data used for the multivariate regression analysis) and Supplemental Figure 1 (Expected Relationship between Changes in Fluorescence (based on AT and GC base pair composition) and Changes in the Threshold Concentration (Ct)) appear online at www.ajtmh.org.

Authors’ addresses: James M. Colborn, Dengue Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, San Juan, PR 00920, E-mail: fue6@cdc.hhs.gov. Brian D. Byrd, College of Health and Human Sciences, Western Carolina University, Cullowhee, NC 28723, E-mail: bdbyrd@wcu.edu. Ousmane A. Koita, Faculty of Science and Technology, University of Bamako, BP E 3206, Bamako, Mali, E-mails: oakoita@yahoo.com and oakoita@ml.refer.org. Donald J. Krogstad, Departments of Tropical Medicine and Medicine, and the Center for Infectious Diseases, Tulane University Health Sciences Center, 1430 Tulane Avenue, SL 17, J. Bennett Johnston Building, New Orleans, LA 70112, E-mail: krogstad@tulane.edu.

Acknowledgments: We thank Fawaz Mzayek for assistance with the statistical analysis; Fran Krogstad for cultivation of P. falciparum; the American Type Culture Collection for providing the Anopheles gambiae G3 strain contributed by Mark Q. Benedict; the Sequencing Core of the Tulane Center for Gene Therapy for sequencing cloned block 2 regions of msp1 from K1, MAD20 and hybrid MAD20/RO33 P. falciparum parasites; Young Hong, Kenneth Swan, Maria Morales, Tunika Okatcha, and Haiyan Deng for thoughtful reviews of the manuscript; and Mark Lawson (Bio-Rad) for permission to cite data from their recent survey about the use of SYBR Green for real-time PCR to estimate copy number.

Financial support: These studies were supported in part by a cooperative agreement from the Emerging Infectious Diseases Program of the Centers for Disease Control and Prevention (CDC) (CCU/UR3 418652). Graduate stipends for James M. Colborn and Brian D. Byrd were supported by the Tulane/CDC Vector-borne Infectious Disease Training Fellowship (CDC Cooperative Agreement T01/CCT622308) and by the Emerging Infectious Diseases Program of the CDC (CCU/ UR3 418652 and U01 CI 000211).

REFERENCES

  • 1

    Schneeberger C, Speiser P, Kury F, Zeillinger R, 1995. Quantitative detection of reverse transcriptase-PCR products by means of a novel and sensitive DNA stain. PCR Methods Appl 4 :234–238.

    • Search Google Scholar
    • Export Citation
  • 2

    Bustin SA, 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25 :169–193.

    • Search Google Scholar
    • Export Citation
  • 3

    Zipper H, Brunner H, Bernhagen J, Vitzthum F, 2004. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Res 32 :e103.

    • Search Google Scholar
    • Export Citation
  • 4

    Zipper H, Buta C, Lammle K, Brunner H, Bernhagen J, Vitzthum F, 2003. Mechanisms underlying the impact of humic acids on DNA quantification by SYBR Green I and consequences for the analysis of soils and aquatic sediments. Nucleic Acids Res 31 :e39.

    • Search Google Scholar
    • Export Citation
  • 5

    Vitzthum F, Geiger G, Bisswanger H, Brunner H, Bernhagen J, 1999. A quantitative fluorescence-based microplate assay for the determination of double-stranded DNA using SYBR Green I and a standard ultraviolet transilluminator gel imaging system. Anal Biochem 276 :59–64.

    • Search Google Scholar
    • Export Citation
  • 6

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193 :673–675.

  • 7

    Campbell CC, Spencer HC, Chin W, Collins WE, 1980. Adaptation of cultured Plasmodium falciparum to the intact squirrel monkey (Saimiri sciureus). Trans R Soc Trop Med Hyg 74 :548–549.

    • Search Google Scholar
    • Export Citation
  • 8

    Teklehaimanot A, Nguyen-Dinh P, Collins WE, Barber AM, Campbell CC, 1985. Evaluation of sporontocidal compounds using Plasmodium falciparum gametocytes produced in vitro. Am J Trop Med Hyg 34 :429–434.

    • Search Google Scholar
    • Export Citation
  • 9

    Mabuchi T, Nishikawa S, 1990. Selective staining with two fluorochromes of DNA fragments on gels depending on their AT content. Nucleic Acids Res 18 :7461–7462.

    • Search Google Scholar
    • Export Citation
  • 10

    Molecular Probes Product Information, (Revision July 1, 2005). OliGreen ssDNA Quantitation Reagent and Kits. Available at: http://probes.invitrogen.com/media/pis/mp07582.pdf.

  • 11

    Rosen S, Skaletsky H, 2000. Primer3 on the WWW for General Users and for Biologist Programmers. Krawetz SA, Misener S, eds. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Totowa, NJ: Humana Press, 365–386.

  • 12

    Blazquez S, Zimmer C, Guigon G, Olivo-Marin JC, Guillen N, Labruyere E, 2006. Human tumor necrosis factor is a chemo-attractant for the parasite Entamoeba histolytica. Infect Immun 74 :1407–1411.

    • Search Google Scholar
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