Performance Evaluation of DBS DNA Extraction Methods in PCR Based Newborn Screening Assays
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1 Performance Evaluation of DBS DNA Extraction Methods in PCR Based Newborn Screening Assays 2011 NBS and Genetic Testing Symposium Suzanne Cordovado, Ph.D. Team Lead, Molecular Quality Improvement Program Newborn Screening and Molecular Biology Branch, Division of Laboratory Sciences NCEH, CDC 9th November 2011 National Center for Environmental Health Centers for Disease Control and Prevention
2 DBS DNA Extraction Study Goals Determine utility of DNA quantitation methods with DBS DNA Examine performance of extracted DNA in assays used in NBS laboratories Evaluate the performance of DNA extracted from 10 year old DBS stored in different conditions Conduct first collaborative study of the NBS Molecular Network
3 Evaluation of DBS DNA Extraction Methods Newborn DBS 5 day DBS, no humidity 6 month DBS -20 C humidity 6 month DBS, no humidity DNA Extraction* Column Purification Magnetic Bead Magnetic Bead Automatic Lysis Quantification qpcr (RNaseP) NanoDrop PicoGreen * DNA was extracted from one 3mm punch
4 DBS DNA Quantitation Methods: PicoGreen, NanoDrop and qpcr Average Concentration ng/ul day 6 mo -20 C 6 mo PicoGreen qpcr (RNaseP) NanoDrop 5 day 6 mo -20 C 6 mo 5 day 6 mo -20 C 6 mo 5 day 6 mo -20 C 6 mo Qiagen Column Magnetic Bead Magnetic Bead Automatic Lysis 5 Day DBS were not quantitated using PicoGreen due to limited DNA
5 Average DNA Yield (ng) from each Extraction Method Extraction method Sample PicoGreen qpcr gdna NanoDrop 5 day N/A Column 6 mo -20 C mo day N/A 242 1,025 Mag Bead 6 mo -20 C mo ,001 5 day N/A Mag Bead 6 mo -20 C ,731 Auto 6 mo ,901 5 day N/A 206 1,265 Lysis 6 mo -20 C ,328 6 mo ,354
6 Average Concentration ng/ul DBS DNA Quantitation qpcr: Using Different Standard Curve Materials PicoGreen qpcr gdna qpcr Lym DNA qpcr pdna 5 day 6 mo -20 C 6 mo Qiagen Column Extraction
7 Evaluation of DBS DNA Extraction Methods Newborn DBS 5 day DBS, no humidity 6 month DBS -20 C humidity 6 month DBS, no humidity DNA Extraction* Column Purification Magnetic Bead Magnetic Bead Automatic Lysis Quantification qpcr (RNaseP) NanoDrop PicoGreen xtag CF 39 Luminex ARMS PCR GALT InPlex CF Hologic RFLP PCR Hemoglobin ß TREC qpcr SCID * DNA was extracted from one 3mm punch
8 DBS Extracted DNA Performance in Molecular NBS Assays xtag CF 39 kit v2 Failures (%) ARMS GALT PCR Failures (%) Inplex CF Failures (%) PCR-RFLP HbB Failures (%) qpcr-trec # TREC (Cq) Age/Storage Col Mag MagA Lys 5 day 0 10 (50) mo -20 C 0 16 (80) 6 (30) 0 6 mo 1 (5) 10 (50) 7 (35) 0 5 day 0 1 (5) mo -20 C (100) 0 6 mo (100) 0 5 day 0 (5) 17 (85) 2 (10) 6 mo -20 C 4 (20) 4 (20) 20 (100) 4 (20) 6 mo (55) 0 5 day (5) 6 mo -20 C mo day 362 (31.6) 342 (31.6) 252 (31.9) 242 (32.2) 6 mo -20 C 319 (31.8) 433 (31.2) 175 (32.7) 244 (32.2) 6 mo 365 (31.4) 444 (31.0) 204 (32.3) 448 (31.0) Assay failures denoted in red and reduced TREC amplification denoted in blue.
9 Study Conclusions - Part 1 DBS DNA quantitation with the NanoDrop overestimates quantity and is not suitable for DBS DNA qpcr performed with different standard curve sources does not perform the same and care should be taken when comparing yields LYM DNA and pdna source fold & 0.87 fold lower than gdna respectively Column based and lysis DNA extractions performed consistently better in all assays
10 Evaluation of PCR Amplification in DBS Newborn DBS 5 day DBS, no humidity 10 year DBS -20 C, humidity 10 year DBS, no humidity DNA Extraction Column Purification Magnetic Bead Magnetic Bead Automatic Lysis Quantification qpcr (RNaseP) 510 bp (1 ng DNA) 1140 bp (2ng DNA) 3000 bp (3 ng DNA) 5620 bp (4 ng DNA)
11 DBS PCR Amplification Results Age/ Storage Extraction 5 Day (N=5) 10 Year -20 C (N=20) 10 Year (N=20) 510 bp Failures (%) 1140 bp Failures (%) 3000 bp Failures (%) 5620 bp Failures (%) Col Mag 3 (60) 0 1 (20) 0 MagA 1 (20) 0 1 (20) 0 Lys Col 1 (5) 1 (5) 0 0 Mag 1 (5) 0 1 (5) 0 MagA 1 (5) 0 12 (60) 7 (35) Lys (5) 2 (10) Col (5) 20 (100) Mag 3 (15) 1 (5) 20 (100) 20 (100) MagA 1 (5) 1 (5) 15 (75) 20 (100) Lys 1 (5) 0 20 (100) 20 (100)
12 Study Conclusions - Part 2 Storage conditions of archival DBS does not affect amplification of smaller fragments ( 1.1 kb) Storage conditions of archival DBS affects amplification of larger fragments ( 3 kb) conditions: 3 kb fragments amplified from DBS extracted using a column method -20 C conditions: 5.6 kb fragment routinely amplified from DBS for all extraction methods
13 A Successful First Collaborative Study Mike Glass Greg Olin Mei Baker Gary Hoffman Greg Kopish Michelle Caggana Carlos A. Saavedra-Matiz Fred Lorey Martin Kharrazi Anne Comeau Jacalyn Gerstel-Thompson Rachel Lee Leslie Covarrubias Susan Tanksley Suzanne Cordovado Miyono Hendrix Laura Hancock Stanimila Nikolova Sean Mochal Daniel Turner Francis Lee
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15 Ladder S4 S4 S4 S4 NTC Ladder S4 S5 S6 S7 S8 S NTC Amplification of 5.6 kb Product 5 day and 10 yr -20 C DBS 10 kb 6 kb 4 kb 5 day/ 10 kb 6 kb 4 kb Col Mag MagA Lys 10 yr/-20c
16 Ladder NTC Amplification of 1.1, 1.9 and 3 kb Products 10 yr DBS 4 kb 3 kb 2 kb 1 kb Col Mag MagA Lys Col Col 10 yr/
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