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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

Developing a Novel Assay forHigh Resolution Melting analysis

Scanning “YFG”

Jason McKinney

Scientist

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

New primers

Original primers

getting started1
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)
design software
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
primer design big picture
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.
primer design
Primer design
  • Flanking primers
    • Allow 10-12 bases of flanking sequence
primer design1
Primer design
  • Overlapping primers
    • Try whole exon scanning first
    • Use internal primers if necessary
primer design specific issues
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)
summary
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