Reports Materials and methods
DNA amplification was performed in 50 mM Tris-HCl, pH 8.5 (25°C), 3 mM MgCl2, 500 µg/mL bovine serum albumin, 0.5 µM of each primer, 0.2 mM of each deoxyribonucleoside triphosphate and 0.2 U of TaqDNA polymerase per 5-µL sample, unless otherwise stated. Human genomic DNA (denatured for 1 min by boiling) or purified amplification product was used as DNA template. Purified amplification product was obtained by phenol /chloroform extraction and ethanol precipitation (17), followed by removal of primers by repeated washing through a Centricon 30 microcon- centrator (Amicon, Beverly, MA, USA). Template concentrations were determined by absorbance at 260 nm. A260
of templates were greater than 1.7. Primers were synthesized by standard
/A280
phosphoramidite chemistry (Gene Assembler Plus; Pharmacia Biotech, Piscataway, NJ, USA). SYBR Green I was obtained from Molecular Probes (Eugene, OR, USA). The β-actin primers and fluorescein/rhodamine dual probe were obtained from Perkin- Elmer (Norwalk, CT, USA). Te human β-globin primers
ratios
fluorophore). Te β-actin hydrolysis probe and the β-globin hybridization probes were used at 0.2 µM each. Figure 1 schemati- cally compares the differences between the three fluorescence monitoring techniques: dsDNA-specific dyes, hydrolysis probes and hybridization probes. Amplification samples of 5 µL were loaded into glass capillary tubes (1.02 mm
RS42/KM29 (536 bp) and PC03/PC04 (110 bp) have been described previously (19). Te single-labeled probes 5´-CAAACA- G ACACCATG GTGCACCT- GACTCCTGAGGA- fluorescein-3´and 5´-Cy5- AAGTCTGCCGTTACTGC- CCTGTGGGGCAAG- phosphate-3´ were synthesized using a fluorescein phosphor- amidite (Glen Research, Sterling, VA, USA), a Cy5 phosphoramidite (Pharmacia Biotech) and a chemical phosphorylation reagent (Glen Research). Tese adjacent probes hybridize internal to the PC03/ PC04 β-globin primer pair on the same DNA strand and are separated by one base pair. Probes were purified by reverse-phase C-18 high pressure liquid chromatography, and homogeneity was checked by polyacryl- amide gel electrophoresis and absorbance (A260
and the absorbance maximum of the
o.d., 0.56 mm i.d.) and sealed. Te tubes were cleaned with optical-grade methanol and then loaded into the carousel of a fluorescence temperature cycler described elsewhere (23). For continuous monitoring of a single sample, the carousel was positioned at maximal fluorescence and signals acquired every 200 ms. For multiple tubes, signals were obtained once each cycle by sequentially positioning the carousel at each tube for 100 ms.
Results
Figure 1 illustrates the three different fluorescence techniques used for continuous monitoring of DNA amplification. Figures 2, 3 and 4 demonstrate the application of these techniques for initial template quantification by fluorescence monitoring once each cycle. In Figure 2, the fluores- cence of the dsDNA-specific dye SYBR Green I is followed. A 107
–108 range of
initial template concentration can be discerned. When the data are normalized as the percent maximal fluorescence of each tube, 100 initial copies are clearly separated from 10 copies. However, the difference between 1 and 10 copies is marginal, and no difference is observed between 0 and 1 average copies per tube. Nonspe- cific detection of undesired products aſter many cycles is a limitation of fluorescence monitoring of dsDNA. Specific fluorescence monitoring can be
obtained with sequence-specific fluorescent probes. In Figure 3, amplification is monitored using a dual-labeled hydrolysis probe. Te fluorescence signal is expressed as a ratio of fluorescein to rhodamine f luorescence. Signal generation with 5´-exonuclease probes is dependent not only on DNA synthesis, but requires hybrid- ization and hydrolysis between the fluoro- phores of the dual-labeled probe. Hydrolysis reduces quenching of fluorescein, and the fluorescence ratio of fluorescein-to-rho- damine emission increases. Whereas the fluorescence from dsDNA-specific dyes plateaus with excess cycling, the signal from hydrolysis probes continues to increase aſter many cycles. Even though no net product is being synthesized, probe hybridization and hydrolysis continue to occur. In contrast to dsDNA dyes, no fluorescence signal is generated in the absence of template. In Figure 4, amplification is monitored
using adjacent hybridization probes and is expressed as a ratio of Cy5 to fluorescein fluorescence. One of the probes is labeled 3´ with fluorescein, and the other probe is labeled 5´ with Cy5. When hybridized to accumulating product, the probes are separated by a 1-bp gap, and the Cy5-to-
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