TABLE 1.

Experimental design and results from the factorial design experiment with the two PCR reaction mixtures based on AmpliTaq Gold and rTtha

PCR mixtureFactorP value by ANOVAbExperimental designPCR results
CTΔRnFactor levelConcnMean CTMean ΔRn
AmpliTaq GoldMgCl20.0000.000High3.5 mM23.11.7
Low1.5 mM36.20.2
Center2.5 mM24.61.2
Axial high4.7 mM22.31.9
Axial low0.1 mM40.00.0
Probe0.1920.050High125 nM30.61.1
Low75 nM28.60.8
Center100 nM26.21.2
Axial high160 nM22.52.1
Axial low40 nM24.50.6
Primer0.2600.089High1,200 nM30.90.9
Low600 nM28.31.0
Center900 nM26.31.1
Axial high1,600 nM23.31.4
Axial low185 nM23.71.2
AmpliTaq Gold0.2180.536High0.07 U/μl29.11.0
Low0.03 U/μl30.10.9
Center0.05 U/μl27.41.1
Axial high0.10 U/μl23.61.4
Axial low0.02 U/μl24.51.2
DNA0.0000.474High5.0 × 10−8 g27.41.0
Low5.0 × 10−10 g31.90.9
Central5.0 × 10−9 g26.21.2
Axial high2.0 × 10−6 g15.71.2
Axial low2.0 × 10−11 g31.51.1
rTthMgCl20.2350.082High3.5 mM24.55.5
Low1.5 mM35.00.5
Center2.5 mM22.35.7
Axial high4.7 mM21.97.5
Axial low0.1 mM36.80.1
Probe0.6820.415High125 nM31.32.8
Low75 nM28.22.8
Center100 nM23.25.5
Axial high160 nM22.44.1
Axial low40 nM24.12.3
Primer0.6810.204High1,200 nM28.13.2
Low600 nM31.42.5
Center900 nM23.45.0
Axial high1,600 nM22.35.8
Axial low185 nM22.08.6
rTth0.7580.769High0.035 U/μl29.63.2
Low0.015 U/μl29.82.4
Central0.025 U/μl23.65.8
Axial high0.043 U/μl23.13.6
Axial low0.007 U/μl21.52.5
DNA0.0030.015High5.0 × 10−8 g27.13.4
Low5.0 × 10−10 g32.42.3
Central5.0 × 10−9 g23.35.1
Axial high2.0 × 10−6 g14.59.1
Axial low2.0 × 10−11 g30.53.8
  • a The different factors and their concentrations are given. The experiment with the AmpliTaq Gold mixture used a basic 25 factorial design with 2-by-5 axial points and one observation at the center point, giving a total of 86 observations. The experiment with the rTth mixture used a basic 25 factorial design augmented with 2-by-5 axial points and five observations at the center point, giving a total of 47 observations.

  • b ANOVA, one-way analysis of variance for main effects in studying the significance of the different factors. Significant factor interactions are not presented here but are given in the text (see Results and Discussion).