Strand Displacement Amplification Example

DESCRIPTION:

Strand Displacement Amplification (SDA) is a method for amplifying DNA that was invented and developed at the Becton Dickinson Research Center. SDA uses two enzymes; namely Hinc II which nicks double stranded target DNA at a specific location and Exo-Klenow which bumps off the existing strand and generates a new strand. This results in exponential multiplication of the number of target pieces of DNA.

This example uses a face-centered cube design which has 3 experimental factors and one response. A face-centered cube design is similar to a central composite design except that the star points have alpha=1 so there are just three levels for each factor. This design has 18 runs including 4 center points.

In this research experiment, the amounts of the two enzymes and the incubation time are varied in order to see if the amount of the more expensive enzyme, Hinc II, can be reduced. The standard operating conditions at the time of this experiment were 2 hours of incubation at 37 degrees C using 150 units of Hinc II and 5 units of Exo-Klenow. This form of the assay produces a colorimetric response, and two responses are recorded; namely a visual score and an optical density reading. It is desired to reduce enzyme levels, thereby reducing the cost of the amplification while maintaining the signal at an easily readable level.

See the chapter on response surface methods in the documentation for more information.

ARGUMENTS:

Hinc.II
an experimental factor specifying the amount of the enzyme Hinc II.
Exo.Klenow
an experimental factor specifying the amount of the enzyme Exo-Klenow.
time
an experimental factor specifying the length of the incubation time.
vis
a response variable, a visual score indicating how strong the colorimetric response is. Higher values are better.
opt.dens
a response variable, the measured value of the optical density of the final color produced by the assay. Higher values are better.

SOURCE:

Experimental data provided by W. Keating of the Becton Dickinson Research Center, Research Triangle Park, NC. Used by permission of Becton Dickinson.

EXAMPLES:

# This design is already available in S-PLUS under  
# the name sda.df. The following commands were used to th 
# However, you could create the design as follows: 
sda.design<-rsm.design(3,alpha=1,factor.names= 
     list(Hinc.II=c(50,150),Exo.Klenow=c(1,5), 
     time=c(2,3))) 
sda.vis <- c(1.5,2.5,1.5,3,2,3.5,2.5,2.5,1.5,3,2.5, 
           2.5,2.5,3.5,3,3.5,2.5,2.5) 
sda.od<-c(1045,2324,700,4300,2862,7266,3502,9687, 
        756,5088,3728,3794,2901,3664,3932,6336, 
        2888,5087)/100 
sda.df <- cbind(sda.design,opt.dens=sda.od,vis=sda.vis) 
# Sample analysis of the response opt.dens.  
# Analysis of the response vis is similar. 
sda.od.rsm <- rsm.lm(sda.df) 
sda.od.rsm 
summary(sda.od.rsm) 
pareto(sda.od.rsm) 
contour(sda.od.rsm,levels=c(20,30,40,50,60), 
      vary=list(Hinc.II='v',time='v')) 
surface(sda.od.rsm,vary=list(Hinc.II='v',time='v')) 
contour(sda.od.rsm,vary=list(Hinc.II='v', 
     Exo.Klenow='v',time=3)) 
plot(sda.od.rsm)