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

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

3. Reliable sequence ?? Original primers New primers

4. Exon locations

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

6. Design software Logistical issuesLogistical issues –Display sequence, regions of interest, primer locations, fragment sizes, etc. Primer designPrimer design –Several packages personal preferencepersonal preference –Experience with design software, ease of use, features –“Tuning” design software Takes time, trial and errorTakes time, trial and error “Tuned” design software is INVALUABLE!“Tuned” design software is INVALUABLE! DO NOT RELY TOO MUCH ON SOFTWAREDO 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 scanningLogical scheme for efficient scanning Minimum # of primer sets, maximum coverageMinimum # of primer sets, maximum coverage –You can always re-design later, … primers are cheap! Let the sequence dictate where primers will be locatedLet the sequence dictate where primers will be located –Difficult to place primers exactly where YOU want them.

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

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

13. Primer design – Specific Issues Predicted Tm’sPredicted Tm’s –Estimations AT BEST –Means of designing similar primers Cross complimentarities (real vs. theoretical)Cross complimentarities (real vs. theoretical) –Hetero- and Homo-dimers “False priming” (real vs. theoretical)“False priming” (real vs. theoretical) –Gene of interest (minimum); Genome (BLAST) 5’ v. 3’ stability, free energy values5’ v. 3’ stability, free energy values Minimum primer length (  17 bases)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 HomeworkDo Your Homework –Specifics about YFG Design SoftwareDesign Software –Ease of use, features, ONLY A TOOL Primer Design – Big PicturePrimer Design – Big Picture –Logical design, let the sequence lead you Primer Design – Specific IssuesPrimer 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 workRemember, they’re just primers, don’t take them too personally, re-design rather than waste time trying to make “bad” primers work