C17: Helicase Dependent Amplification of HPRT1 at Different...

In chipPCR: Toolkit of Helper Functions to Pre-Process Amplification Data

Description

A Helicase Dependent Amplification (HDA) of HPRT1 (Homo sapiens hypoxanthine phosphoribosyltransferase 1) was performed at different temperatures in the 'VideoScan' Platform 2.0 (similar to Roediger et al. (2013)). The HDA was performed at 55, 60 and 65 degrees Celsius. The optimal temperature for a HDA is circa 65 degrees Celsius. Lower temperatures will affect the slope and plateau of the HDA amplification curve.

Usage

data(C17)

Format

A data frame with 125 observations on the following 5 variables.

C17.t

Elapsed time during HDA in seconds.

C17.cycle

a numeric vector

C17.T55

Time-dependent fluorescence at 55 degrees Celsius

C17.T60

Time-dependent fluorescence at 60 degrees Celsius

C17.T65

Time-dependent fluorescence at 65 degrees Celsius

Details

To perform an isothermal amplification in 'VideoScan' 2.0, standard conditions for the IsoAmp(R) III Universal tHDA Kit (Biohelix) were used. The reaction was composed of 12.5 micro L buffer A containing 1.25 micro L 10x reaction buffer, 150 nM primer (forward and reverse), 0.75 micro L template (synthetic) and A. bidest which was covered with 50 micro L mineral oil. The primer sequences for HPRT1 were taken from Roediger et al. (2013). Preincubation: This mixture was incubated for 2 min at 95 degree. Celsius and immediately placed on ice. 12.5 micro L of reaction buffer B which was composed of 1.25 micro L 10x buffer, 40 mM NaCl, 5 mM MgSO4, 1.75 micro L dNTPs, 0.2 x EvaGreen, 1 micro L Enzyme mix and A. bidest. The fluorescence measurement in 'VideoScan' 'HCU' started directly after adding buffer B at 55, 60 or 65 degrees Celsius and revealed optimal conditions for the amplification when using 60 or 65 degrees Celsius. Temperature profile (after Preincubation): - 60 seconds at 65 degrees Celsius - 11 seconds at 55 degrees Celsius && Measurement

Source

Claudia Deutschmann & Stefan Roediger, BTU Cottbus - Senftenberg, Senftenberg, Germany

References

A Highly Versatile Microscope Imaging Technology Platform for the Multiplex Real-Time Detection of Biomolecules and Autoimmune Antibodies. S. Roediger, P. Schierack, A. Boehm, J. Nitschke, I. Berger, U. Froemmel, C. Schmidt, M. Ruhland, I. Schimke, D. Roggenbuck, W. Lehmann and C. Schroeder. Advances in Biochemical Bioengineering/Biotechnology. 133:33–74, 2013.

Examples

data(C17)
plot(NA, NA, xlim = c(0,5000), ylim = c(0,1.2), xlab = "Time [sec]", 
     ylab = "Fluorescence", 
     main = "Temperature dependency of HDA amplification reactions")
  points(C17[, 1], C17[, 3], type = "b", col = 1, pch = 20)
  points(C17[, 1], C17[, 4], type = "b", col = 2, pch = 20)
  points(C17[, 1], C17[, 5], type = "b", col = 3, pch = 20)
legend(2000, 0.4, c("55 degrees Celsius", "60 degrees Celsius", 
       "65 degrees Celsius"), 
	col = c(1,2,3), pch = rep(20,3))

chipPCR documentation built on March 5, 2021, 9:06 a.m.