Array-based studies for single nucleotide polymorphism (SNP) interrogation and copy number variation using genomic DNA samples are demanding applications. One popular human array, the Genome-Wide Human SNP Array 6.0 from Affymetrix, features 1.8 million genetic markers, including greater than 900,000 SNPs and more than 946,000 probes for detecting copy number variation (1) . Commercially available genomic DNA (gDNA) purification systems are available for isolating human gDNA in formats that vary both in the nature of the DNA capture (affinity membrane, affinity particles or alcohol precipitation) and the volume of the starting sample volume (small volume, ~200–300µl, and large volume, 3–10ml). The purification method can affect the molecular weight of the isolated DNA due to the mechanical action of the purification (Figure 1); however, all of the systems still yield DNA suitable for PCR analysis.
Figure 1. CHEF gel electrophoresis analysis of gDNA isolated from blood. Genomic DNA (0.5µg) was isolated from blood samples in triplicate using each system and analyzed on a 1% agarose gel using the CHEF Mapper® (BioRad). Genomic DNA was stained with ethidium bromide and compared to two molecular weight ladders: M1, ProMega-Markers® Lambda Ladders (Cat.# G3011) and M2, Lambda DNA/HindIII Markers (Cat.# G1711).
Here we compare the new Promega cellulose-based gDNA purification systems to traditional silica and precipitation-based chemistries by isolating gDNA from human blood or buccal swabs for microarray analysis. DNA purity and concentration were evaluated by absorbance spectroscopy. Samples that met the minimum concentration requirements were submitted to the Genomics Core Facility at the Medical College of Wisconsin for analysis using the Affymetrix Genome-Wide Human SNP Array 6.0. All samples generated results that exceeded the existing quality control standards for the core facility.
Genomic DNA Purification Systems
Maxwell® HT 96 gDNA Blood Isolation System
The Maxwell® HT 96 gDNA Blood Isolation System uses a novel cellulose-based purification method to isolate gDNA from up to 200µl of whole blood (2) . The binding matrix and chemistry allow samples to be processed in a microcentrifuge without using ethanol or isopropanol in any of the purification solutions. Genomic DNA is recovered in as little as 30µl of nuclease-free water.
Maxwell® 16 LEV Blood DNA and Buccal Swab LEV DNA Purification Kits
The Maxwell® 16 LEV Blood DNA and Maxwell® 16 Buccal Swab LEV DNA Purification Kits are used with the Maxwell® 16 Instrument to provide an easy method for efficient, automated purification using the MagnaCel™ cellulose magnetic resin (3,4). The Maxwell® 16 Instrument is supplied with preprogrammed purification procedures and is designed for use with the predispensed reagent cartridges for maximum simplicity and convenience. After an initial proteinase K treatment, the instrument can process up to 16 samples in approximately 45 minutes. The purified DNA can be used directly in a variety of downstream applications including qPCR and HLA testing. The system is recommended for up to 400µl of blood or 1–2 buccal swabs. Genomic DNA can be eluted in volumes as small as 50µl.
QIAamp® DNA Blood Mini Kit
QIAamp® DNA Blood Mini Kit (Qiagen) provides silica-membrane-based DNA purification. Mini kits are designed for processing up to 200μl of human whole blood, and DNA is eluted in a final volume of 50–200µl.
Wizard® Genomic DNA Purification Kit
The Wizard® Genomic DNA Purification Kit provides a simple, solution-based method for isolating DNA from white blood cells, tissue culture cells, animal tissue, plant tissue, yeast or Gram-positive and Gram-negative bacteria. DNA purified with this system is suitable for many applications, including amplification, digestion with restriction endonucleases and membrane hybridizations. This protocol uses alcohol precipitation of the gDNA for recovery and can be scaled up from a starting volume of 50µl of sample.
ReliaPrep™ Large Volume HT gDNA Isolation System
The ReliaPrep™ Large Volume HT gDNA Isolation System isolates gDNA from blood samples ranging from 3–10ml in a scalable format. The MagnaCel™ cellulose chemistry eliminates tedious centrifugation steps as well as the use of hazardous chemicals, which are inherent in precipitation-based chemistries. The system has been automated on the Tecan Freedom EVO® liquid-handling workstation, allowing walkaway purification of gDNA from 32 or 64 samples of 3–10ml of whole blood in 4.5 or 6 hours, respectively, regardless of sample storage or shipping conditions (5,6). For low-throughput isolation of gDNA from less than 32 samples at one time, the ReliaPrep™ LV 32 HSM Instrument can be used in a manual mode, where the user performs the pipetting functions.