PCR Assay Optimization
Optimize PCR by
Understanding Relative Primer Efficiency |
This example
shows how Visual OMP can be used to optimize a PCR assay
developed for detection of Cryptosporidium parvum in water*.
The primers are designed to amplify a region of the 18S
rRNA gene. |
| |
|
|
 |
|
Visual OMP is used to optimize
PCR assay conditions for maximal unbiased amplification efficiency.
The assay was simulated, and OMP identified an optimal annealing
temperature of 53.7ºC, matching the experimentally determined
result of 54ºC under the initial conditions: [Mg+2]=2mM,
[Na+]=0.05M, [target strand concentration]=1 pM, [primer
AWA995F]=0.2µM, [primer AWA1206R]=0.2µM |
| |
|
|
 |
|
Further in
silico investigation of PCR conditions reveals that performing
the PCR under lower magnesium concentration (new [Mg+2] =
1 mM) requires the annealing temperature to be lowered to
52ºC for maximal unbiased PCR amplification efficiency.
[Mg+2]=1mM, [Na+]=0.05M, [target strand
concentration]=1 pM, [primer AWA995F]=0.2µM, [primer
AWA1206R]=0.2µM |
| |
|
|
 |
|
In this third
scenario, primer binding efficiency was equalized by adjusting
the relative primer concentrations. Since the forward primer
binds more weakly to its template strand, a higher concentration
is needed to ensure equal amplification. This analysis can
be performed before any resources are spent in lengthy empirical
PCR optimization protocols. [Mg+2]=2mM,
[Na+]=0.05M, [target strand concentration]=1 pM, [primer
AWA995F]=0.5µM, [primer AWA1206R]=0.1µM |
| |
| *
Rochelle P.A. et al.: Comparison of primers and optimization
of PCR conditions for detection of Cryptosporidium parvum
and Giardia lamblia in water. (1997). Applied and Environmental
Microbiology, v. 63(1), pp106-114
Home
|
Products |
Services | Science
| Support |
About Us | Site Map
|
Contact Us
© 2006 DNA Software, Inc.
All Rights Reserved.
|
|
|
