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Developing a Novel Assay for High Resolution Melting analysis

Developing a Novel Assay for High Resolution Melting analysis. Scanning “YFG”. Jason McKinney Scientist. Getting started …. Reliable, verifiable sequence of YFG Confirm sequence from multiple sources Collaborating with an “expert” * Exon locations. Reliable sequence ??. New primers.

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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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