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)