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© 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
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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
© Copyright 2009 by the American Association for Clinical Chemistry
IntroductionIntroduction 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
© Copyright 2009 by the American Association for Clinical Chemistry
IntroductionIntroduction 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
© Copyright 2009 by the American Association for Clinical Chemistry
Introduction (cont)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
© Copyright 2009 by the American Association for Clinical Chemistry
QuestionQuestion Why is it important that UGT1A1 (TA)n genotyping assays be validated for the (TA)5 and (TA)8 repeat alleles?
© Copyright 2009 by the American Association for Clinical Chemistry© 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 MethodsMaterials and Methods
© Copyright 2009 by the American Association for Clinical Chemistry
Materials and Methods (cont)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
© Copyright 2009 by the American Association for Clinical Chemistry
Materials and Methods (cont)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)
© Copyright 2009 by the American Association for Clinical Chemistry
QuestionQuestion What structure does a snapback primer form after PCR and how does this structure reveal the (TA)n repeat genotype?
© Copyright 2009 by the American Association for Clinical Chemistry
ResultsResults 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
© Copyright 2009 by the American Association for Clinical Chemistry
Results (cont)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
© Copyright 2009 by the American Association for Clinical Chemistry
Results (cont)Results (cont)
Actual Genotype n Miscalled Genotype n(TA)5/(TA)6 6
(TA)5/(TA)7 7 (TA)5/(TA)6 1
(TA)5/(TA)8 2
(TA)6/(TA)6 22 (TA)5/(TA)6 1
(TA)6/(TA)7 34 (TA)6/(TA)6 4 (TA)7/(TA)7 1
(TA)6/(TA)8 7 (TA)6/(TA)7 3(TA)7/(TA)7 1(TA)7/(TA)8 1
(TA)7/(TA)7 15 (TA)6/(TA)7 1
(TA)7/(TA)8 7 (TA)7/(TA)7 3Total 100 16
Small amplicon genotyping on a capillary-based instrument
84% accuracy compared to fragment analysis after correction for fragment analysis error
© Copyright 2009 by the American Association for Clinical Chemistry
Results (cont)Results (cont)
Actual Genotype n Miscalled Genotype n
(TA)5/(TA)6 5 0
(TA)5/(TA)7 6 (TA)6/(TA)7 2
(TA)5/(TA)8 2 (TA)5/(TA)7 1
(TA)6/(TA)6 21 (TA)5/(TA)7 1 (TA)6/(TA)7 1
(TA)6/(TA)7 33 (TA)7/(TA)7 3
(TA)6/(TA)8 7 (TA)6/(TA)7 1(TA)7/(TA)7 3
(TA)7/(TA)7 15 (TA)6/(TA)8 3(TA)7/(TA)8 3
(TA)7/(TA)8 6 0Total 95 18
Snapback primer genotyping on a plate-based instrument 100% accuracy on
capillary-based instrument fell to 81% on a plate-based instrument
© Copyright 2009 by the American Association for Clinical Chemistry
QuestionQuestion Why is genotyping accuracy dependent on the
instrument?
© Copyright 2009 by the American Association for Clinical Chemistry
ConclusionsConclusions 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
© Copyright 2009 by the American Association for Clinical Chemistry
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