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.

1. Integrated DNA Technologies
Finding the Right Primers:
Using NCBI for RT-PCR Primer Design
Adam Clore, PhD

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

7. 1. Type in the gene of interest

8. 2. Select Genes: Gene-centered information

9. 3. Choose the species you are interested in.

10. All known transcript variants are displayed.

11. 4. Select “reference sequence details” for information on each transcript variant.

12. Details about a specific transcript variant.

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

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

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

23. NCBI SNP Database
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