Specializing in the design and troubleshooting of qPCR, mutagenesis, and cloning experiments at IDT, Scientific Applications Specialist, Adam Clore PhD, gave this presentation about the intelligent selection of PCR primers. Topics include identifying transcript variants for your target of interest and selecting primers that amplify only specific transcripts. Adam also discussed the growing SNP database and the potential impact of SNPs on qPCR data, how to locate any SNPs that fall in your target amplicon, and the use of free NCBI and IDT tools to help you avoid them.
2. Considerations When Designing RT-PCR Assays
Physical Properties of the Primers
and Probe
Transcript Variants
SNPs
Adam Clore, PhD
Finding the Right Primers
3. What is a Transcript Variant and Why Should You Care?
Genes with different
combinations of exons
Alternatively spliced transcripts
produce different isoforms of
proteins
95% of human genes have
alternate splicing
Many cancers and some genetic
diseases are caused by transcript
variants
Adam Clore, PhD
Finding the Right Primers
Gene
Exons
Introns
Differential
Splicing
Transcript
Variants
4. Role of Variants in Normal Gene Expression
Doublesex gene in D. melanogaster influences sex development by
alternatively splicing the 3 exons of the gene:
Pre-mRNA
Female variant
Male variant
Adam Clore, PhD
Finding the Right Primers
5. Role of Transcript Variants in Disease
correlating with cancer:
CD44
Wilms’ tumor gene WT1
BRCA1
MDM2
FGFR
Adam Clore, PhD
Finding the Right Primers
6. NCBI Gene GUI
Use to see all known transcript variants in
the RefSeq database and how they differ.
Adam Clore, PhD
Finding the Right Primers
13. Options for Real Time PCR Design
PrimeTime® Predesigned qPCR Assay Library
For Human, Mouse, and Rat
All designs are screened for crosstalk between variants
http://www.idtdna.com/order/predesignedassay.aspx?source=scitools
RealTime Design Tool
Real-time design for sequences entered by Ref Seq number or sequences
pasted in manually
http://www.idtdna.com/scitools/Applications/RealTimePCR/
Adam Clore, PhD
Finding the Right Primers
14.
15.
16. Exon Naming…
NCBI has had no official convention
An example of how it’s often done
Variant #2
Variant #1
1 2 3 4 5
1 2 3 4 5 6
Adam Clore, PhD
Finding the Right Primers
17. Exon Naming…
What NCBI (and IDT) are moving to:
Variant #2
Variant #1
1 2 3 4 7 8
1 5 6 7 8
Adam Clore, PhD
Finding the Right Primers
18.
19.
20.
21.
22. What is a SNP and Why Do I Care?
Single nucleotide
polymorphism a single letter
change in the genetic code
There are an approximately
56,000,000 SNPs in the human
genome
16,000,000 are in gene introns
and exons
Most are silent mutations
Some are clinically relevant
Adam Clore, PhD
Finding the Right Primers
33. Summary
IDT RT-PCR Design Tools
PrimeTime® Predesign aPCR Assay Tool
For Human, Mouse, or Rat
Identifies transcript variants and avoids SNPs in all Pre-designed Assays
RealTime Design Tool
Can design for any sequences submitted by user
User needs to check for transcript variants and SNPs
Adam Clore, PhD
Finding the Right Primers