Biomedical Engineering Reference
In-Depth Information
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Figure 6.3
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be transferred to a SYBR Green I real-time format, but new assays are simple to design
and this is recommended, especially if the legacy assay turns out to be suboptimal. Primer
design for real-time PCR largely follows the same recommendations as for end-point
PCR. The main difference is that the amplicon size is best kept below 250 bp, ideally
between 75 and 150 bp. It is important to select primer pairs that do not show significant
potential for self-dimerization, because primer dimers also contribute to the SYBR Green
I quantification signal. Primers should also be specific for the desired target, particularly
avoiding 3
hybridization to reduce mispriming and linear amplification events. There
are a variety of software options, with Primer3 (http://primer3.sourceforge.net/) being
freely available, or more sophisticated options such as Beacon Designer (Premier Biosoft).
In addition, many companies that provide oligos also offer assay design services (e.g.
www.sial.com/designmyprobe from Sigma-Genosys). An alternative to designing a new
assay is to identify a previously designed, validated set of primers and reaction conditions.
The best sources of primers and probes specifically designed for RT-qPCR assays are public
primer and probe databases such as RTPrimerDB (http://medgen.ugent.be/rtprimerdb/),
PrimerBank (http://pga.mgh.harvard.edu/primerbank/index.html), or Real Time PCR
Primer Sets (http://www.realtimeprimers.org/). RTPrimerDB lists validated qPCR assays
submitted by researchers for the commonly used chemistries and includes all the
information required to understand the purpose of an assay and to implement them in an
experiment.
Although the primers alone determine the specificity of product detection, information
regarding product size and population is easily determined from a melt curve analysis. Melt
curves are a means of providing identification of amplified products and distinguishing them
from primer dimers and other small amplification artifacts. The melting temperature (
T
m
)
of DNA is defined as the temperature at which half of the DNA helical structure is lost.
