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 Typing

2. Maxwell® 16 System

9. Conclusion

www.promega.com

Automated DNA Purification from Buccal Swabs for qPCR and HLA Typing assaysusing the Maxwell® 16 LEV Blood KitRebecca 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 November 2010

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mbp

1,000750500

300

150

50

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

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).

ng/µl 260/280 Yield (µg)

1 swab 136.05 1.90 6.80

1 swab 102.16 1.82 5.11

1 swab 174.96 1.89 8.75

1 swab 128.62 1.96 6.43

1 swab 148.91 1.95 7.45

1 swab 112.10 1.71 5.61

1 swab 130.38 1.78 6.52

AVERAGE 133.3 1.86 6.67

Method1. Collect samples with a standard buccal swab collection

procedure.2. Add dried swab head to Clearing Column/Microtube

assembly.3. In a separate tube, mix 300µl Lysis Buffer +30µl

Proteinase K for each sample.4. Add 330µl of Lysis Buffer/ProK to swab head in Clearing

Column/Microtube assembly. Close tube and vortex for 10 seconds.

5. Incubate for 20 minutes at 56°C.6. After incubation, centrifuge for 2 minutes at maximum

speed. 7. Remove the Clearing Column with swab head and

discard.8. Add flow-through to Well #1 of the Maxwell 16 LEV

Blood cartridge.

• Process using Maxwell® 16 in LEV Research Mode using the LEV Blood method.

Clearing Column/Microtube Assembly

Materials Needed Microcentrifuge56˚C incubator1.5ml microtubes

Cat. #V1231 recommended Clearing Columns, Cat. #Z387AMaxwell® 16 LEV Blood Kit, Cat. #AS1290

The Maxwell® 16 Instrument

The Maxwell® LEV format prefilled reagentcartridge

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.

ng/µl 260/280 260/230

2 swabs 121.85 1.81 1.52

2 swabs 209.01 1.82 1.64

2 swabs 276.45 1.87 1.74

2 swabs 274.13 1.8 1.55

2 swabs 122.16 1.8 1.58

2 swabs 173.5 1.91 1.82

2 swabs 235.9 1.81 1.69

AVERAGE 201.86 1.83 1.65

Customer Quantification Data(Blood Center of Wisconsin data)

ng/µl 260/280 260/230

1 swab 97.98 1.88 1.94

1 swab 190.01 1.73 1.4

1 swab 65.06 1.82 1.67

1 swab 194.52 1.76 1.41

1 swab 251.36 1.96 2.09

1 swab 121.88 1.88 1.78

1 swab 136.33 1.88 1.96

1 swab 147.6 1.91 2.15

AVERAGE 150.59 1.85 1.8

bp

1,000750

500

300

150

50

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M

Donor 2

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.

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.

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 thatmay 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.

Promega Quantification Data from One and Two Swabs

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.