ABSTRACT
The success of the polymerase chain reaction
(PCR) is highly dependent on primer design.
Commonly used primer design programs
rely upon a core set of parameters such as
melting temperature, primer length, GC
content, and complementarity to optimize
the PCR product, but weight those
parameters to differing degrees, as well as
include other parameters for PCR specific
tasks. An analysis of these design algorithms
,and other available PCR primer analysis
software, was conducted to find the best
means of predicting non-specific PCR
products in a laboratory environment using
Saccharomyces cerevisiae deletion strains.
Results here show that there is no web-based
program that is well-suited to the task of
post-design PCR analysis for non-specific
annealing and secondary structure in the
context of the whole genome. A brute force
technique was employed using the National
Center for Biotechnology Information’s
(NCBI’s) Megablast and Zuker’s mfold to
correlate primer sequence similarities and
secondary structure predictions in a full
genome context, with inconclusive results.
The ultimate conclusion is that there is a
need for a user-friendly, post-design PCR
analysis program to accurately predict nonspecific
hybridization events that impair
PCR amplification.