Developing a Novel Assay forDeveloping a Novel Assay forHigh Resolution Melting analysisHigh Resolution Melting analysis

Scanning “YFG”Scanning “YFG”

Jason McKinneyJason McKinney

ScientistScientist

Getting started …Getting started …

• Reliable, verifiable sequence of YFGReliable, verifiable sequence of YFG– Confirm sequence from multiple sourcesConfirm sequence from multiple sources– Collaborating with an “expert” *Collaborating with an “expert” *

• Exon locationsExon locations

Reliable sequence ??Reliable sequence ??

Original primersOriginal primers

New primersNew primers

Exon locationsExon locations

Getting started …Getting started …

• Identify Common or Rare mutations, Identify Common or Rare mutations, polymorphisms, location in YFGpolymorphisms, location in YFG– Useful tools for assay Useful tools for assay

optimization/validationoptimization/validation

• What needs to be scanned?What needs to be scanned?– 5’ UTR, 3’ UTR, splice sequence regions5’ UTR, 3’ UTR, splice sequence regions

• Samples * (collaboration with expert)Samples * (collaboration with expert)

Design softwareDesign software

• Logistical issuesLogistical issues– Display sequence, regions of interest, primer locations, Display sequence, regions of interest, primer locations,

fragment sizes, etc.fragment sizes, etc.

• Primer designPrimer design– Several packagesSeveral packages

• personal preferencepersonal preference

– Experience with design software, ease of use, featuresExperience with design software, ease of use, features– ““Tuning” design softwareTuning” 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

Design Software – Design Software – Sequence viewSequence view

Design Software – Design Software – primer viewprimer view

Design software - Design software - Gene ViewGene View

Primer design – Big picturePrimer design – Big picture

• Logical scheme for efficient scanningLogical scheme for efficient scanning• Minimum # of primer sets, maximum Minimum # of primer sets, maximum

coveragecoverage– You can always re-design later, … primers You can always re-design later, … primers

are cheap!are cheap!

• Let the sequence dictate where primers Let the sequence dictate where primers will be locatedwill be located– Difficult to place primers exactly where Difficult to place primers exactly where

YOU want them.YOU want them.

Primer designPrimer design

• Flanking primersFlanking primers– Allow 10-12 bases of flanking sequenceAllow 10-12 bases of flanking sequence

• Overlapping primersOverlapping primers– Try whole exon scanning firstTry whole exon scanning first– Use internal primers if necessaryUse internal primers if necessary

Primer designPrimer design

Primer design – Specific IssuesPrimer design – Specific Issues• Predicted Tm’sPredicted Tm’s

– Estimations AT BESTEstimations AT BEST– Means of designing Means of designing similarsimilar primers primers

• Cross complimentarities Cross complimentarities (real vs. theoretical)(real vs. theoretical)– Hetero- and Homo-dimersHetero- and Homo-dimers

• ““False priming” False priming” (real vs. theoretical)(real vs. theoretical)– Gene of interest (minimum); Genome (BLAST)Gene of interest (minimum); Genome (BLAST)

• 5’ v. 3’ stability, free energy values5’ v. 3’ stability, free energy values• Minimum primer length (Minimum primer length ( 17 bases) 17 bases)

Primer design – good locationPrimer design – good location

Primer design – AT richPrimer design – AT rich

Primer design – Primer design – “bad” on paper“bad” on paper

Primer design – too easyPrimer design – too easy

SummarySummary• Do Your HomeworkDo Your Homework

– Specifics about Specifics about YFGYFG

• Design SoftwareDesign Software– Ease of use, features, ONLY A TOOLEase of use, features, ONLY A TOOL

• Primer Design – Big PicturePrimer Design – Big Picture– Logical design, let the sequence lead youLogical design, let the sequence lead you

• Primer Design – Specific IssuesPrimer Design – Specific Issues– Use software to “evaluate” YOUR designs for similar Use software to “evaluate” YOUR designs for similar

characteristics, increase chances for successful primer characteristics, increase chances for successful primer designsdesigns

• Remember, they’re just primers, don’t take them too Remember, they’re just primers, don’t take them too personally, re-design rather than waste time trying to personally, re-design rather than waste time trying to make “bad” primers workmake “bad” primers work