Molecular beacon-based temperature control and automated analyses for improved resolution of melting temperature analysis using SYBR I green chemistry.
[FIGURE 1 OMITTED]
High-resolution Tm analyses were not the original purpose of most gPCR instruments. Temperature variations over the heating block and low numbers of fluorescence measurements during the dissociation step hamper the ability of most instruments to report accurate and precise Tms (9). To improve Tm analyses without acquiring a specialized instrument for the purpose (such as the HR-1[TM] from Idaho Technology), 2 major issues must be resolved, the temperature variations inherent to the heating block must be normalized and more precise Tms be calculated from low-resolution temperature data.
Tm analysis programs that apply a curve-fit method to the data points obtained during melting have been developed for other systems. We present a program that was adapted for the ABI Prism[R] 7000 with SDS v1.2.3, but the principles of this program can be adapted for other systems. In addition, we present the application of a Tm-probe used to control for temperature variations, similar to that applied for a microfluidic platform by Dodge et al. (10).
[FIGURE 2 OMITTED]
Materials and Methods
QPCR AND DISSOCIATION CURVE
We used an ABI Prism 7000 SDS (Applied Biosystems) with a Precision Plate Holder (Applied Biosystems), white Thermo-Fast[R] 96 detection plates (ABgene), and the version 1.2.3 SDS software package (Applied Biosystems). The Platinum SYBR Green gPCR SuperMix UDG (Invitrogen) was used in each of the 25-[micro]L reactions. Unless stated otherwise, reactions were performed with 250 nmol/L of the forward (TCA GGT CAA CAA TAG GAT GAC AAC A) and reverse (CAA TGA GGG TCT ACA CTG GGA ACT) primers and 133 nmol/L of the Tm probe (FAM-TTT TTT T(-TAMRA)TC GGC CGC TCC CCC CCC CCC CCA GCG GCC GA-BHQ2). The passive reference dye ROX was not included. Unless stated otherwise, 3100 copies of a pcDNA2.1 (Invitrogen) TA cloning plasmid containing a HERV-W gag fragment from chromosome 3826 were used as an amplification template (1) (see Fig. 5). Signal-to-noise ratios were calculated as the signal fluorescence during the last cycles divided by the noise fluorescence during the first cycles (11). Reaction efficiencies of the assay in the presence or absence of the Tm-probe were calculated as previously described (1).
[FIGURE 3 OMITTED]
TM PROBE DESIGN
To allow detection in an ABI Prism 7000 simultaneously as SYBR I Green fluorescence, a molecular beacon (12,13) was designed to have a stem structure with a Tm higher than those observed for the target transcript amplicons (85 [degrees]C), for which the SYBR signal is minimal [Web page for tm analysis and generation of Fig. 2 folding http://www.bioinfo.rpi.edu/applications/mfold/dna/forml.cgi (14-16)]. During denaturation, the fluorescence of molecular beacons increases rather than decreases upon melting, allowing the derivative curve of dissociation data to be easily distinguished from that of any SYBR products. To obtain absorption and emission wavelengths appropriate for the instrument, we used a wavelength-shifting molecular beacon design (17). The molecular beacon was triple-labeled with 6-carboxyfluorescein (FAM) in the 5' end, 6-carboxytetramethylrhodamine (TAMRA) attached to the 6th thymidine from FAM, and Black Hole Quencher (BHQ)-2 in the 3' end (Fig. 1). In the hybridized configuration the FAM in the Tm-probe absorbs the 485-nm excitation provided by the instrument. The high-energy state FAM undergoes fluorescence resonance energy transfer (FRET) to TAMRA, which in turn donates its energy through FRET to the BHQ-2. At temperatures >85 [degrees]C the molecular beacon undergoes a conformational change, and the TAMRA will no longer transfer any energy to BHQ-2 because it is no longer in close enough proximity and will thus fluoresce at 580 run. The Tm probe was purchased from MedProbes (Eurogentec).
We used the MATLABTM (The MathWorks) version 184.108.40.20604 with The Optimization Toolbox to write an automated analysis algorithm for data from Sequence Detection Software version 1.2.3 used in conjunction with an ABI Prism 7000. The program, gaussian curve fit analysis of TM (GcTm), was designed to determine Tms of amplicons by fitting gaussian curves to derivative data from dissociation analyses (Fig. 2). The peak of the negative derivative data is automatically selected by taking the values differing from the mean derivative over all temperatures by at least 1.2 SDs. We also designed the program to use the Tm of the Tm-probe to normalize temperatures of amplified products reported from the instrument in each well. The Tm normalization calculation took the Tms, determined by GcTm, of the amplicon minus that of the corresponding Tm-probe plus the average of all the Tm-probes used in that experiment. The program is available for download at http://www.neuro.ki.se/kristensson/tmanalysis.html.
To evaluate the Tm analysis methods we used a plasmid template in 25-[micro]L reactions in all wells of a 96-well plate. The Tms reported by the SDS software over the 96-well plate show a range of 0.6 [degrees]C in reported temperatures (SD 0.19 [degrees]C). Each Tm was reported as 1 of 3 discreet temperatures (Fig. 3). Analysis with the GcTm algorithm of the dissociation data gave Tms with higher resolutions than those reported by the SDS software, based on a gaussian curve fitted to multiple data points (on average 5.25, Fig. 3), but the systematic variations in reported temperatures remained (range 0.66 [degrees]C). To control for the temperature variations over the heating block, a molecular beacon-based Tm-probe was designed (Fig. 1). Gaussian curves fitted to derivative dissociation curve data for the SYBR and TAMRA detectors are shown in Fig. 2. The negative derivative dissociation data for the Tm-probe with the TAMRA detector displays an artifactual peak with changes in SYBR fluorescence, because in our assay SYBR has a broad emission spectrum and fluoresces an order of magnitude more than TAMRA. In the interest of cost-effectiveness, we used the lowest concentration of Tm-probe giving reliable Tms with low variation. The Tms for the Tm-probe calculated with GcTm showed variations over the heating block similar to those of the amplicons (Fig. 3). Correcting the Tms of the amplicons by subtracting those of the Tm-probe (and adding the average Tm of all the Tm-probes) gave a profile over the 96-well plate with no apparent systematic variations with regard to well position (Fig. 3). Variations in reported Tms had an SD of 0.18 [degrees]C for the SYBR alone and an SD of 0.12 [degrees]C when corrected with the Tm-probe (Fig. 4). The Tm-probe showed a higher variation in Tms within wells than the amplicons. In addition, although the TAMRA fluorescence displayed spatial variation similar to that of SYBR I Green, the TAMRA fluorescence fluctuated more. Therefore the Tm-probe showed high accuracy but lower precision. To reduce variations, we set the ABI Prism 7000 to run 2 additional dissociation curve analyses immediately after the primary run. These dissociations were analyzed individually with GcTm and the resulting Tms averaged. The mean of 3 dissociations resulted in an SD of 0.17'C for the SYBR alone and an SD of 0.06'C when corrected with the Tm-probe (Fig. 4). Additional dissociations beyond the 3 described did not further decrease the SDs of the corrected Tms. To illustrate the improved precision and accuracy, we analyzed 4 different plasmids with variations in the target sequences (Fig. 5A). The addition of the Tm-probe allowed the identification of 3 distinct groups of Tms, indicating at least 3 unique sequences that were indistinguishable without correction of amplicon Tms (Fig. 5B). Uncorrected and corrected Tms for each of the 4 plasmid templates are shown in Fig. 5C to demonstrate that sequence variations do not necessarily give Tm differences. Although the Tm-probe described in the present work had no effect on reaction efficiency (data not shown), the fluorescence emitted by FAM, which is not transferred by FRET to TAMRA, increased the background and thereby reduced the signal-to-noise ratio from 9 to 4.5.
[FIGURE 4 OMITTED]
According to the manufacturer's specifications of the ABI Prism 7000 instrument, the temperature uniformity over a 96-well block is [+ or -]0.5 [degrees]C, a feature that limits the obtainable resolution when Tms of amplicons are examined. Using the Tm of a plasmid template as a reporter, we found that higher Tms were reported consistently in the middle of the 96-well reaction plate and that Tms were reported as discreet temperatures (Fig. 3). We wrote an analytical algorithm to extrapolate accurate Tms from multiple data points. Using this algorithm in conjunction with a molecular beacon-based Tm-probe, we improved resolution 3-fold.
[FIGURE 5 OMITTED]
Some caveats should be considered with the use of the proposed Tm analyses. The reason for the observed variations in Tm-probe melting temperatures between wells is not known. However, the tests performed have shown a greater variation in wells flanked by empty wells or in wells at the corners of the plate, possibly a sign of evaporation effects and salt-concentration changes affecting the Tm-probe to a larger extent than the amplicons.
Furthermore, for Tm analyses of multiple sequences in the same reactions, SYBR I Green chemistry may not be optimal and other dyes (such as LCGreen) have been suggested to be more appropriate (18). Although the use of LCGreen can improve the determination of amplicon Tms, it will not eliminate the need for 3 dissociation analyses to be performed, because the primary source of variation stems from the Tm-probe measurements rather than those of the amplicons. SYBR I Green chemistry is adequate for comparison of Tms of amplicons between wells. It should be noted that differing sequences can share the same Tm, as is the case with the plasmids corresponding to the HERV-W gag on chromosomes 5p13 and 12p13.
In conclusion, we report methodological improvements for Tm analyses on an ABI Prism 7000 instrument. By use of the addition of a Tm-probe in combination with a curve-fit method (GcTm) and repeated measurements to determine Tms of amplicons, we have improved Tm precision from [+ or -]0.36 [degrees]C to [+ or -]0.12 [degrees]C (with a 95% confidence interval).
This study was generously supported by the Stanley Medical Research Institute and the Swedish Research Council (project no. K2006-21X-20047-01-3). The authors declare that they have no competing interests. We thank Cecilia Eckervig from MedProbes whose expertise aided the design of the Tm-probe. We also thank Dr. Yuanrong Yao in our laboratory for providing the plasmids used as templates.
Received June 19, 2006; accepted October 19, 2006. Previously published online at DOI: 10.1373/clinchem.2006.075184
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 Nonstandard abbreviations: gPCR, quantitative real-time PCR; Tm, melting temperature; FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; BHQ, Black Hole Quencher-2; FRET, fluorescence resonance energy transfer; GcTm, Gaussian curve fit analysis of Tm.
CHRISTOFFER NELLAKER,  * ULF WaLLGREN  and HAKAN KARLSSON 
 Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
 FLIR Systems AB, Danderyd, Sweden.
* Address correspondence to this author at: Retzius vig 8, 171 77 Stockholm, Sweden. Fax: +468325325; e-mail email@example.com.
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|Title Annotation:||Automation and Analytical Techniques|
|Author:||Nellaker, Christoffer; Wallgren, Ulf; Karlsson, Hakan|
|Date:||Jan 1, 2007|
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