1. Snapback Primer Genotyping of the Gilbert Syndrome UGT1A1 (TA) n Promoter Polymorphism by High-Resolution Melting J.S. Farrar, R.A. Palais, and C.T. Wittwer www.clinchem.org/cgi/content/article/57/9/1303 September 2011 © Copyright 2011 by the American Association for Clinical Chemistry

2. © Copyright 2009 by the American Association for Clinical Chemistry Introduction  Gilbert syndrome  A chronic non-hemolytic unconjugated hyperbilirubinemia  Associated with thymine-adenine (TA) insertions in the UGT1A1 promoter  UGT1A1 promoter genotype also correlates with toxicity induced by the chemotherapeutic drug irinotecan

3. © Copyright 2009 by the American Association for Clinical Chemistry Introduction  UGT1A1 (TA) n promoter polymorphism  Repeat length varies: (TA) 5, (TA) 6, (TA) 7, (TA) 8  Frequency and length of repeats depends on ethnicity  (TA) 5 and (TA) 8 alleles restricted to African descent  Current genotyping methods  Multiple manual steps, multiple labeled probes and/or difficulty genotyping the (TA) 5 and (TA) 8 alleles

4. © Copyright 2009 by the American Association for Clinical Chemistry Introduction (cont)  Snapback primer genotyping  Closed-tube, high-resolution melting method  Uses a saturating double-stranded DNA dye instead of covalently modified primers/probes  Only two standard PCR primers are needed (one with a 5’-addition)  Has previously been applied to single-base variants and small deletions

5. © Copyright 2009 by the American Association for Clinical Chemistry Question  Why is it important that UGT1A1 (TA) n genotyping assays be validated for the (TA) 5 and (TA) 8 repeat alleles?

6. © Copyright 2009 by the American Association for Clinical Chemistry Snapback primer approach for genotyping the UGT1A1 (TA) n promoter polymorphism. A probe complementary to the (TA) 5 repeat is added as a 5’ addition to the forward PCR primer (gray). Asymmetric PCR overproduces a single-stranded DNA product that forms an intra-molecular hairpin. Products with repeats greater than (TA) 5 form bulge loops that progressively destabilize the hairpin structure. Hairpin stability is revealed by high-resolution melting analysis. Materials and Methods

7. © Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods (cont)  100 African American DNA samples  Study population enriched for (TA) 5 and (TA) 8 alleles  New melting analysis method  Plots the local deviation from exponential decay in order to remove background fluorescence  Improves genotype clustering

8. © Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods (cont)  3 different genotyping methods  Fragment analysis (reference method)  Small amplicon melting  Snapback primer genotyping  2 blinded studies  Small amplicon melting and snapback primer genotyping on a capillary-based instrument  Instrument comparison for snapback primer genotyping (capillary vs. plate-based)

9. © Copyright 2009 by the American Association for Clinical Chemistry Question  What structure does a snapback primer form after PCR and how does this structure reveal the (TA) n repeat genotype?

10. © Copyright 2009 by the American Association for Clinical Chemistry Results  Snapback primer genotyping on a capillary-based instrument  99% concordant with genotyping results from fragment analysis  Reanalysis of single discordant sample revealed an error in fragment analysis  100% accuracy after correction for error in fragment

11. © Copyright 2009 by the American Association for Clinical Chemistry Results (cont)  Snapback primer genotyping on a capillary- based instrument  Although the melting temperature differences are small, the absolute temperature precision of the instrument and the shapes of the melting curves enable accurate genotyping

12. © Copyright 2009 by the American Association for Clinical Chemistry Results (cont) Actual GenotypenMiscalled Genotypen (TA) 5 /(TA) 6 6 ­ (TA) 5 /(TA) 7 7(TA) 5 /(TA)61 (TA) 5 /(TA) 8 2 ­ (TA) 6 /(TA) 6 22(TA) 5 /(TA)61 (TA) 6 /(TA) 7 34(TA)6/(TA)64 (TA)7/(TA)71 (TA) 6 /(TA) 8 7(TA)6/(TA)73 (TA)7/(TA)71 (TA)7/(TA)81 (TA) 7 /(TA) 7 15(TA) 6 /(TA) 7 1 (TA) 7 /(TA) 8 7(TA) 7 /(TA) 7 3 Total100 16  Small amplicon genotyping on a capillary-based instrument  84% accuracy compared to fragment analysis after correction for fragment analysis error

13. © Copyright 2009 by the American Association for Clinical Chemistry Results (cont) Actual GenotypenMiscalled Genotypen (TA) 5 /(TA) 6 5­­0 (TA) 5 /(TA) 7 6(TA)6/(TA)72 (TA) 5 /(TA) 8 2(TA) 5 /(TA)71 (TA) 6 /(TA) 6 21(TA)5/(TA)71 (TA)6/(TA)71 33(TA)7/(TA)73 (TA) 6 /(TA) 8 7(TA) 6 /(TA) 7 1 (TA) 7 /(TA) 7 3 15(TA) 6 /(TA) 8 3 (TA) 7 /(TA) 8 3 6­­0 Total95 18  Snapback primer genotyping on a plate-based instrument  100% accuracy on capillary-based instrument fell to 81% on a plate-based instrument

14. © Copyright 2009 by the American Association for Clinical Chemistry Question  Why is genotyping accuracy dependent on the instrument?

15. © Copyright 2009 by the American Association for Clinical Chemistry Conclusions  Instrument and genotyping method are critical for successful genotyping  Plate-based high-resolution melting instrument did not perform as well as capillary-based instrument  Absolute temperature precision of the instrument is critical  Snapback primer genotyping performed better than small amplicon genotyping  Snapback primers can be used to genotype simple sequence repeats in <30 min

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