Developing a novel assay for high resolution melting analysis
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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


Design software sequence view
Design Software – Sequence view




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)




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



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


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