1. Introduction. 3. Method for Automated DNA Extraction from Buccal Swabs. 5.
SSO Typing (URMC data). 4. Concentration of DNA and qPCR Analysis. 8. SSP ...
Automated DNA Purification from Buccal Swabs for qPCR and HLA Typing assays ® using the Maxwell 16 LEV Blood Kit Rebecca Gorshe, MS1, Jennifer Schiller, PhD2, Danielle Meehan, CHT3, and Trista Schagat, PhD1 1Promega Corporation, Madison, WI; 2Blood Center of Wisconsin, Milwaukee, WI; 3University of Rochester Medical Center, Rochester, NY
1. Introduction
8. SSP Typing
4. Concentration of DNA and qPCR Analysis
The Human Leukocyte Antigen (HLA) system is a group of genes that makes up the major histocompatibility complex (MHC). This super locus contains a large number of genes related to immune system function in humans. HLA typing is used to determine whether a tissue or organ donor is a suitable match for a patient. Typically, DNA is extracted from blood or buffy coat and the DNA is analyzed by Sequence Specific Oligonucleotides (SSO) typing or Sequence Specific Priming (SSP) typing. The use of buccal or cheek swabs is an advantageous alternative to a blood draw because of its non-invasiveness and the lack of reliance on the donors white blood cell count. Here we describe an application of the Maxwell® 16 LEV Blood DNA Kit (Cat. #AS1290) for the automated extraction of DNA from buccal swab samples for use with SSO and SSP analysis for HLA typing. We also describe this application for compatibility for qPCR assays.
Promega Quantification Data from One and Two Swabs
Customer Quantification Data (Blood Center of Wisconsin data)
ng/µl
260/280
260/230
1 swab
97.98
1.88
1.94
1.64
1 swab
190.01
1.73
1.4
1.87
1.74
1 swab
65.06
1.82
1.67
274.13
1.8
1.55
1 swab
194.52
1.76
1.41
2 swabs
122.16
1.8
1.58
1 swab
251.36
1.96
2.09
5.61
2 swabs
173.5
1.91
1.82
1 swab
121.88
1.88
1.78
1.78
6.52
2 swabs
235.9
1.81
1.69
1 swab
136.33
1.88
1.96
1.86
6.67
AVERAGE
201.86
1.83
1.65
1 swab
147.6
1.91
2.15
AVERAGE
150.59
1.85
1.8
ng/µl
260/280
260/230
2 swabs
121.85
1.81
1.52
5.11
2 swabs
209.01
1.82
1.89
8.75
2 swabs
276.45
128.62
1.96
6.43
2 swabs
1 swab
148.91
1.95
7.45
1 swab
112.10
1.71
1 swab
130.38
AVERAGE
133.3
ng/µl
260/280
Yield (µg)
1 swab
136.05
1.90
6.80
1 swab
102.16
1.82
1 swab
174.96
1 swab
Data suggest DNA concentration is compatible with multiple HLA assays without the need to concentrate. While two swabs did yield more DNA, the amount was not linear. Variability between samples is normal as the amount of DNA recovered will depend on donor and collection technique. Concentration requirements for the SSP kit tested are 75-125ng/µl.
2.
Maxwell®
November 2010
DRB5 SSP analysis was performed on samples per manufacturer’s instructions for the DRB5 SSP Unitray® System (Invitrogen Cat. #45131-2). Below are the results of 2 individuals that displayed positive allele markers for the DRB5 region. The data shown match previous typings for the 2 individuals. Donor 1
bp
M
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 M
1,000 750 500 300
16 System
150 50
Donor 2
The Plexor® HY System (Cat. #DC1000) is a real-time PCR assay to determine the concentration of total human DNA and male human DNA simultaneously in one reaction. The kit contains an internal PCR control (IPC) to test for false-negative results that may occur in the presence of PCR inhibitors and a melt curve function to confirm that the correct product was amplified. All buccal swab samples were successfully amplified with no detectable PCR inhibitors.
The Maxwell® LEV format prefilled reagent cartridge
The Maxwell® 16 Instrument
M
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
M
bp 1,000 750 500
5. SSO Typing (URMC data)
300 150
The Maxwell® 16 Instrument is a compact, plug-n-play system that uses prefilled, ready to use reagent cartridges for consistent purification of nucleic acids from a variety of sample types. The Maxwell® uses paramagnetic particles (PMPs) that are coated with silica or cellulose. The large surface area of the PMPs allows for a high binding capacity. Chaotropic lysis buffer disrupts tissues and cells while a series of alcohol washes remove unwanted debris. The nucleic acid is then eluted in a low salt (or water) buffer. The low elution volume (LEV) format (shown) allows elution of nucleic acid in as little as 30μl.
50
The control band of 796bp is present in all allelic lanes (1-14) and the no DNA control lane (15) is negative. According to the DRB5 SSP UniTray® product literature, donor 1 is predicted to carry the DRB5 allele 0202 due to the products in lanes 11 and 12 (185bp and 200bp, respectively). Donor 2 is predicted to carry the DRB5 allele 010101/0202 due to the products in lanes 1 and 2 (115bp and 260bp, respectively).
3. Method for Automated DNA Extraction from Buccal Swabs Materials Needed Microcentrifuge 56˚C incubator 1.5ml microtubes Cat. #V1231 recommended Clearing Columns, Cat. #Z387A Maxwell® 16 LEV Blood Kit, Cat. #AS1290
1. 2. 3. 4.
5. 6. 7. 8.
Method Collect samples with a standard buccal swab collection procedure. Add dried swab head to Clearing Column/Microtube assembly. In a separate tube, mix 300µl Lysis Buffer +30µl Proteinase K for each sample. Add 330µl of Lysis Buffer/ProK to swab head in Clearing Column/Microtube assembly. Close tube and vortex for 10 seconds. Incubate for 20 minutes at 56°C. After incubation, centrifuge for 2 minutes at maximum speed. Remove the Clearing Column with swab head and discard. Add flow-through to Well #1 of the Maxwell 16 LEV Blood cartridge. •
9. Conclusion
We have developed an application with the Maxwell® 16 LEV Blood Kit (Cat. #AS1290) for the automated extraction of DNA from buccal swabs. The DNA extracted is compatible with assays such as qPCR and SSO/SSP analysis for HLA typing. Preliminary customer data (Blood Center of Wisconsin) also shows compatibility of Maxwell® 16 extracted DNA with an additional method for HLA typing, Sequence Based Testing (data not shown). The use of an automated extraction system allows the user consistent recovery of DNA from the samples that is sufficient for accurate allele determination for HLA typing. In addition, DNA can be isolated in ~1 hour with minimal hands on time, allowing for quick turnaround when time may be critical for HLA typing.
Process using Maxwell® 16 in LEV Research Mode using the LEV Blood method.
Clearing Column/Microtube Assembly
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