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Developing a Novel Assay for High Resolution Melting analysis

This project focuses on creating a robust assay for high-resolution melting (HRM) analysis of the "Your Favorite Gene" (YFG). The aim is to reliably confirm and verify the sequence of YFG from multiple sources and to assist in identifying common and rare mutations, along with polymorphisms. The study emphasizes the importance of primer design, exploring different tools and software to optimize and validate the assay. It also addresses logistical issues and the significance of comprehensive primer placement to achieve maximum coverage of the target sequence.

aphrodite-stephenson
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Developing a Novel Assay for High Resolution Melting analysis

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  1. Developing a Novel Assay forHigh Resolution Melting analysis Scanning “YFG” Jason McKinney Scientist

  2. Getting started … • Reliable, verifiable sequence of YFG • Confirm sequence from multiple sources • Collaborating with an “expert” * • Exon locations

  3. Reliable sequence ?? New primers Original primers

  4. Exon locations

  5. Getting started … • Identify Common or Rare mutations, polymorphisms, location in YFG • Useful tools for assay optimization/validation • What needs to be scanned? • 5’ UTR, 3’ UTR, splice sequence regions • Samples * (collaboration with expert)

  6. Design software • Logistical issues • Display sequence, regions of interest, primer locations, fragment sizes, etc. • Primer design • Several packages • personal preference • Experience with design software, ease of use, features • “Tuning” design software • Takes time, trial and error • “Tuned” design software is INVALUABLE! • DO NOT RELY TOO MUCH ON SOFTWARE

  7. Design Software – Sequence view

  8. Design Software – primer view

  9. Design software - Gene View

  10. Primer design – Big picture • Logical scheme for efficient scanning • Minimum # of primer sets, maximum coverage • You can always re-design later, … primers are cheap! • Let the sequence dictate where primers will be located • Difficult to place primers exactly where YOU want them.

  11. Primer design • Flanking primers • Allow 10-12 bases of flanking sequence

  12. Primer design • Overlapping primers • Try whole exon scanning first • Use internal primers if necessary

  13. Primer design – Specific Issues • Predicted Tm’s • Estimations AT BEST • Means of designing similar primers • Cross complimentarities (real vs. theoretical) • Hetero- and Homo-dimers • “False priming” (real vs. theoretical) • Gene of interest (minimum); Genome (BLAST) • 5’ v. 3’ stability, free energy values • Minimum primer length ( 17 bases)

  14. Primer design – good location

  15. Primer design – AT rich

  16. Primer design – “bad” on paper

  17. Primer design – too easy

  18. Summary • Do Your Homework • Specifics about YFG • Design Software • Ease of use, features, ONLY A TOOL • Primer Design – Big Picture • Logical design, let the sequence lead you • Primer Design – Specific Issues • Use software to “evaluate” YOUR designs for similar characteristics, increase chances for successful primer designs • Remember, they’re just primers, don’t take them too personally, re-design rather than waste time trying to make “bad” primers work

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