© Copyright 2009 by the American Association for Clinical Chemistry Enrichment and Detection of Rare Alleles by Means of Snapback Primers and Rapid-Cycle PCR L. Zhou, R.A. Palais, G.D. Smith, D. Anderson, L.R. Rowe, and C.T. Wittwer May © Copyright 2010 by the American Association for Clinical Chemistry Journal Club

© Copyright 2009 by the American Association for Clinical Chemistry Introduction  In early and post treatment stages of cancer Mutation allele fraction may be low (<10%) Mutations may be undetectable by sequencing  Current enrichment methods have limitations Scorpion primers (complex) TaqMan (fluorescent labels) PNA and LNA (specially modified oligos) ARMS (false positives)

© Copyright 2009 by the American Association for Clinical Chemistry Introduction (cont)  Snapback primers Closed-tube genotyping method Use saturating dyes instead of covalent fluorescent labels No special modifications Only 2 primers are required One primer has a 5’-tail that hybridizes to its extension product

© Copyright 2009 by the American Association for Clinical Chemistry Questions  What is a snapback primer?  What structure does a snapback primer form after PCR?

© Copyright 2009 by the American Association for Clinical Chemistry Figure 1. PCR and melting analysis of snapback primers. Snapback primers are standard oligonucleotides that include as a probe element a 5’-tail that is complementary to the extension product of the primer. Both full-length amplicons and intramolecular hairpins are formed after PCR. Melting of the probe element of the snapback hairpin provides targeted genotyping, whereas high-resolution analysis of amplicon melting optionally detects variants anywhere within the PCR product.

© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods  Mechanism of enrichment Mismatch the rare allele to the probe element Use polymerase without 5’-exonuclease activity Completely matched wild type allele blocks extension Mismatched rare allele amplifies more efficiently

© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods (cont)  Snapback enrichment factors Probe element length and Tm Extension temperature Extension time Mg ++ concentration

© Copyright 2009 by the American Association for Clinical Chemistry Materials and Methods (cont)  Control human cell lines BRAF p.600E homozygote EGFR p.delE746-A750 homozygote  Clinical Samples 47 patients with thyroid tumors 8 patients with lung tumors

© Copyright 2009 by the American Association for Clinical Chemistry Questions  How do snapback primers enrich minority alleles?  What factors influence enrichment?

© Copyright 2009 by the American Association for Clinical Chemistry Figure 2. Mechanism of enrichment using snapback primers. When the PCR conditions are carefully chosen, the polymerase unfolds and extends the destabilized mutant hairpins, but wild type extension is blocked, resulting in enrichment of the mutant allele.

© Copyright 2009 by the American Association for Clinical Chemistry Results  Factors that increase minority allele enrichment with snapback primers Longer snapback probe elements with higher Tms Lower extension temperature Shorter extension time Lower Mg ++ concentration

© Copyright 2009 by the American Association for Clinical Chemistry Figure 3. The effect of snapback probe element length and Tm on allele enrichment. In (A) the 9 bp probe element (Tm=64°C), shows balanced peaks without apparent allele enrichment. In (B), the 13 bp probe element (Tm=68°C) shows some preference for the mismatched allele. In (C), the 17 bp probe element (Tm=74°C) definitely enriches the mismatched allele. A B C

© Copyright 2009 by the American Association for Clinical Chemistry Figure 4. The effect of extension temperature on allele enrichment with snapback primers. As the extension temperature is decreased from 76ºC (left panel) to 70ºC (right panel), the perfectly matched wild type allele in the heterozygote appears to disappear. A/G G A 95°C 0s 63°C 0s 76°C 5s 95°C 0s 63°C 0s 70°C 5s

© Copyright 2009 by the American Association for Clinical Chemistry Figure 5. The effect of extension time on snapback primer allele enrichment. The extension times were varied between 0 and 20 s at a free Mg ++ concentration of 1.2 mM. The shorter the extension time, the higher the apparent mutation fraction. The initial minor allele percentages were either 1% (circles) or 0.1% (triangles).

© Copyright 2009 by the American Association for Clinical Chemistry Figure 6. The effect of Mg ++ concentration on snapback primer allele enrichment. The free Mg++ concentration was varied between 0.8 mM and 2.2 mM, with the extension temperature at 70°C for 0 s. The lower the Mg ++ concentration, the higher the apparent mutation fraction. The initial minor allele percentages were either 1% (circles) or 0.1% (triangles).

© Copyright 2009 by the American Association for Clinical Chemistry Results  Sensitivity of rare allele detection 0.1% for BRAF p.600E 0.02% for EGFR p.delE746-A750  Snapback primers and HybProbes correlate R 2 = 0.93 More mutations identified with snapback primers

© Copyright 2009 by the American Association for Clinical Chemistry Question  Do the results support the proposed mechanism of snapback primer enrichment?

© Copyright 2009 by the American Association for Clinical Chemistry Figure 7. Analysis of thyroid nodules for BRAF p.V600E with both dual hybridization probes and snapback primers after enrichment. In panel A (left), standard curves for dual hybridization probes (HybProbes ®, triangles) and snapback primers (circles), correlate the apparent mutation fraction to the actual mutation fraction. In panel B (right), the standard curves of each method are used to compare the actual mutation fractions of 91 thyroid samples using both HybProbe and snapback primer analysis.

© Copyright 2009 by the American Association for Clinical Chemistry Discussion  Snapback primer genotyping can be modified to enrich minor alleles in tumor samples High probe element Tm relative to extension temperature Short extension times Low Mg ++ concentration

© Copyright 2009 by the American Association for Clinical Chemistry Discussion (cont)  Rapid cycle PCR enables snapback primer enrichment PCR efficiency of mutation mismatch > wild type match 70 cycles, although excessive by typical standards, is completed within 25 min