Transcript Commercial DNA Extraction Methods for Dried Blood Spots

Commercial DNA Extraction
Methods for Dried Blood Spots
NBS Molecular Training Class
June 28 – 30, 2011
Suzanne Cordovado, PhD
Molecular Quality Improvement Program, CDC
National Center for Environmental Health
Centers for Disease Control and Prevention
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
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
Lysis DNA Extraction with a
Pre-Wash cont.
Step 1:
Add cell lysis solution to break open
cell.
Remove supernate to wash away
contaminants including heme &
other proteins
Step 2:
Add DNA elution solution AND heat
to remove DNA from the DBS.
Selecting a DNA Extraction
Method for DBS
Lysis DNA
Extraction
Without Pre-Wash
Pros
Most
inexpensive
Fast
Cons
Crude prep
Fragmented
DNA
Low DNA
concentration
With Pre-Wash
Pros
Removes
some
contaminants
Cons
Not highly
purified
Inexpensive
Fragmented
DNA
Fast
Fixed elution vol
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
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.
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.
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.
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.
Selecting a DNA Extraction
Method for DBS
Highly Purified DBS
DNA Extraction
Column Based
Method
Pros
High quality DNA
High yield
Adjustable
elution volume
Magnetic Bead Based
Method
Cons
Labor
Intensive
Expensive
Pros
High quality
DNA
High yield
Automatable
Adjustable
elution volume
Cons
Labor
Intensive
Expensive
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
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
DBS DNA Extraction Evaluation
Decision Tree Partners
Public Health Collaborators:
California, Massachusetts, New York, Texas,
Washington, Wisconsin and CDC
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)
NBS Assay Performance Results
DNA extraction
Age
6 month
Magnetic Bead (Omega)
6 month
Manual
3 day
6 month
Magnetic Bead (Omega)
6 month
Robotic
3 day
6 month
QiaAmp Micro Columns
6 month
(Qiagen)
3 day
6 month
5 Prime (Formerly
Gentra Generations)
6 month
3 day
Storage
-20C, humidity
control
RT, no humidity
control
RT, no humidity
control
-20C, humidity
control
RT, no humidity
control
RT, no humidity
control
-20C, humidity
control
RT, no humidity
control
RT, no humidity
control
-20C, humidity
control
RT, no humidity
control
RT, no humidity
control
xTAG CF 39 kit v2
(Luminex)
Arms multiplex GALT PCR
(in-house developed)
Inplex CF
(Hologic)
PCR-RFLP Hemoglobin B
(in-house developed)
Failures (%)
Failures (%)
Failures (%)
Failures (%)
Quantitative PCR - TREC
(in-house developed)
Mean # TRECs/ul blood
(Median Cq)
16 (80%)
0 (0%)
4 (20%)
11 (55%)
433 (31.2)
10 (50%)
0 (0%)
0 (0%)
2 (10%)
444 (31.0)
10 (50%)
1 (5%)
1 (5%)
0 (0%)
342 (31.6)
6 (30%)
20 (100%)
20 (100%)
4 (20%)
175 (32.7)
7 (35%)
20 (100%)
11 (55%)
3 (15%)
204 (32.3)
0 (0%)
0 (0%)
17 (85%)
1 (5%)
252 (31.9)
1 (5%)
0 (0%)
4 (20%)
0 (0%)
319 (31.8)
1 (5%)
0 (0%)
0 (0%)
0 (0%)
365 (31.4)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
362 (31.6)
4 (20%)
0 (0%)
4 (20%)
0 (0%)
244 (32.2)
0 (0%)
0 (0%)
0 (0%)
0 (0%)
448 (31.0)
0 (0%)
0 (0%)
2 (10%)
1 (5%)
242 (32.2)
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.
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
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)