1 / 18

Commercial DNA Extraction Methods for Dried Blood Spots

NBS Molecular Training Class June 28 – 30, 2011. Commercial DNA Extraction Methods for Dried Blood Spots. Suzanne Cordovado, PhD Molecular Quality Improvement Program, CDC. National Center for Environmental Health. Centers for Disease Control and Prevention.

tom
Download Presentation

Commercial DNA Extraction Methods for Dried Blood Spots

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. NBS Molecular Training ClassJune 28 – 30, 2011 Commercial DNA Extraction Methods for Dried Blood Spots Suzanne Cordovado, PhD Molecular Quality Improvement Program, CDC National Center for Environmental Health Centers for Disease Control and Prevention

  2. Selecting a DNA Extraction Method for DBS • Lysis Extraction Method • With or without initial washes • Highly Purified Extraction Methods • Column Based Purification • Magnetic Bead Based Purification

  3. Lysis DNA Extraction with a Pre-Wash Step 1: Add cell lysis solution to break open cells Remove supernate which contains contaminants including heme & other proteins

  4. Lysis DNA Extraction with a Pre-Wash cont. Step 1: Add cell lysis solution to break open cell. Remove supernateto wash away contaminants including heme & other proteins Step 2: Add DNA elution solution AND heat to remove DNA from the DBS.

  5. Lysis DNA Extraction Without Pre-Wash With Pre-Wash Pros Cons Pros Cons Selecting a DNA Extraction Method for DBS Crude prep Fragmented DNA Low DNA concentration Removes some contaminants Inexpensive Fast Most inexpensive Fast Not highly purified Fragmented DNA Fixed elution vol

  6. Selecting a DNA Extraction Method for DBS • Lysis Extraction Method • With or without initial washes • Highly Purified Extraction Methods • Column Based Purification • Magnetic Bead Based Purification

  7. Column DNA Extraction Step 1: Add cell lysis solution to break open cell and heat to remove everything from the DBS. Add binding buffer so the DNA will stick to the column matrix. Remove entire mixture to be put in column.

  8. Column DNA Extraction Step 1: Add cell lysis solution to break open cell and heat to remove everything from the DBS. Add binding buffer so the DNA will stick to the column matrix. Remove entire mixture to be put in column. Step 2: Add lysed cell mixture to filter matrix.

  9. Column DNA Extraction Step 1: Add cell lysis solution to break open cell and heat to remove everything from the DBS. Add binding buffer so the DNA will stick to the column matrix. Remove entire mixture to be put in column. Step 2: Add lysed cell mixture to filter matrix. Centrifuge column to push proteins through the matrix keeping DNA in column.

  10. Column DNA Extraction Step 1: Add cell lysis solution to break open cell and heat to remove everything from the DBS. Add binding buffer so the DNA will stick to the column matrix. Remove entire mixture to be put in column. Step 2: Add lysed cell mixture to filter matrix. Centrifuge column to push proteins through the matrix keeping DNA in column. Step 3: Add DNA elution reagent to detach from filter and centrifuge into tube.

  11. Highly Purified DBS DNA Extraction Column Based Method Magnetic Bead Based Method Pros Cons Pros Cons Selecting a DNA Extraction Method for DBS Labor Intensive Expensive High quality DNA High yield Automatable Adjustable elution volume High quality DNA High yield Adjustable elution volume Labor Intensive Expensive

  12. Potential PCR Inhibitors • Inherent in blood or may be introduced during collection (anticoagulants) • Hemoglobin • Immunoglobulin G • Lactoferrin • Heparin • Impact should be determined for each assay • Assay robustness • Assay sensitivity

  13. DBS DNA Extraction Evaluation Decision Tree • Evaluate DNA extraction methods using fresh DBS (3 days) and 6 month old DBS • 4 Extraction methods used: • 5 Prime Lysis • Qiagen Micro Column • Omega-BioTek Magnetic Bead (by hand) • Omega-BioTek Magnetic Bead with KingFisher robotics

  14. DBS DNA Extraction Evaluation Decision Tree Partners Public Health Collaborators: California, Massachusetts, New York, Texas, Washington, Wisconsin and CDC

  15. DBS DNA Extraction Evaluation Decision Tree cont. Newborn Screening Assays Used for Evaluation: • ARMs multiplex GALT (WI) • xTAG CFTR 39v2 Luminex (MA) • InPlex CFTR Hologic (NY) • PCR-RFLP Hemoglobin B (TX) • Quantitative PCR TREC (CDC)

  16. NBS Assay Performance Results Note: Assay failures are in red – for the multiplex assays, if any part of the assay failed, the run was called failed. Reduced amplification in the TREC assay is in blue.

  17. Extraction Method Details • Omega Bio-Tek Magnetic Bead • E-Z 96 MagBind Kit (cat # 1427-01) • $1.78/sample • www.omegabioteck.com • Qiagen Column • QIAamp DNA Blood Micro Kit (cat # 56304) • $3.70/sample • www.qiagen.com • 5 Prime Lysis • 5 Prime Ready PCR DNA (cat # 159992, 159994) • $0.69/sample • www.qiagen.com

  18. Discussion of DNA Extraction Methods Used by Ongoing State Public Health Labs Discussion Panel: Mei Baker (WI), Carlos A. Saavedra-Matiz (NY), Rachel Lee (TX), Anne Comeau (MA)

More Related