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Characteristics and Tendencies of Pull-up peaks

Characteristics and Tendencies of Pull-up peaks. Carrie Rowland Forensic Bioinformatic Services, Inc. Dayton, Ohio www.bioforensics.com. An Overview : Where are we going. Mechanism – How does dye separation occur Characterize Pull-up Tendencies – are we surprised ?? Conclusions.

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Characteristics and Tendencies of Pull-up peaks

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  1. Characteristics and Tendencies of Pull-up peaks Carrie Rowland Forensic Bioinformatic Services, Inc. Dayton, Ohio www.bioforensics.com

  2. An Overview: Where are we going • Mechanism – How does dye separation occur • Characterize Pull-up • Tendencies – are we surprised ?? • Conclusions

  3. Mechanism: What is pull-up ??? Matrix failure – Inability of the detection instrument to properly resolve the dye colors used to label STR amplicons due to spectral overlap. Pull-up or bleed through occurs.

  4. Mechanism: How the dyes are separated

  5. AmpFlSTR® Profiler Plus™ Kit available from PE Biosystems (Foster City, CA) 200 bp 300 bp 400 bp 100 bp Size Separation D3 vWA FGA 5-FAM (blue) JOE (green) A D8 D21 D18 Color Separation D5 D13 D7 NED (yellow) ROX (red) GS500-internal lane standard 9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction An Example Forensic STR Multiplex Kit

  6. Filter

  7. How a matrix works in GeneScan® Amt. spectral overlap in ea dye into Blue

  8. 5 -FAM JOE NED GeneScan® matrix file table ROX

  9. Characterization: Arriving at pull-up Criteria for pull-up: 1. Data collection point was ± bp relative to primary peak 2. Not in a +4 and -4 position 3. Not a spike or blob 4. Peaks were at least 50 RFUs

  10. Characterization: What pull-up looks like Positive control 9947A

  11. Research data set (Thanks Keith) • Data set consisted of 36 distinct reference samples • 33 or the 36 samples exhibited pull-up • 55 total pull-up peaks

  12. Characterization: what the data set showed • 82% below the recommended saturation level of 4000 RFUs • 97.2% of pull-up peaks spawned by a primary peak at the D8 loci (51 of 54 peaks). • Minimum primary peak ht. to give rise to pull-up – 93 RFUs • Maximum ht. of a pull-up peak – 202 RFUs

  13. CHARACTERISTICS OF THE PRIMARY PEAK 82% 18% 56%

  14. Characterization: Primary peak vs. Pulled up peak

  15. CONCLUSIONS ● Pull-up is common – look for it ● Can and will appear below saturation level ● Most commonly spawned by peaks in green (D8) , then blue (D3). ● Most common pull-up up peak is off ladder ●

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