Finding the Right Primers: Using NCBI for RT-PCR Primer Design
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The document discusses considerations for designing RT-PCR assays, highlighting the importance of understanding transcript variants and SNPs in genetic research. It details how to utilize the NCBI database for identifying transcript variants and provides tools for real-time PCR design. Additionally, it mentions the prevalence of alternative splicing in human genes and its implications in diseases like cancer.
Finding the Right Primers: Using NCBI for RT-PCR Primer Design
- 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