II. Product Components
III. Materials to be Supplied by the User
IV. Protocol for Extraction of DNA from Whole Blood
V. Protocol for GenomePlex Whole Genome Amplification from Whole Blood
VI. Quantification of Amplified Products
VII. Purification of Amplified Products
Whole blood is a common source of material when performing genetic analysis. Many times genomic DNA from samples can have low yield. This quantity can hinder the researcher's ability to perform downstream analysis. This method describes amplifying nanogram amounts of starting genomic DNA with little or no detectable bias resulting in microgram quantities. In addition, the protocol provides a simple and convenient method to isolate pure genomic DNA from fresh or aged whole blood, amplify the genomic DNA, and rapidly purify the PCR amplified products. The methods described are completed using the GenElute Blood Genomic DNA Kit, GenomePlex WGA kit, and GenElute PCR Clean-up Kit.
GenomePlex is a Whole Genome Amplification (WGA) method that allows the researcher to generate a representative, approximate 300 to 1000-fold amplification of genomic DNA. The amplification yield is dependent on the purity and amount of starting material. The kit utilizes a proprietary amplification technology based upon random fragmentation of genomic DNA and conversion of the resulting small fragments to PCR-amplifiable OmniPlex Library molecules flanked by universal priming sites. The OmniPlex li brary is then PCR amplified using universal oligonucleotide primers and a limited number of cycles. This technology maintains the genetic representation with concordance >99.8% in genotyping results from comparing genomic DNA and GenomePlex amplified DNA1. In addition to SNP genotyping, downstream applications also include performing TaqMan assays and BeadArray analysis.
Note: The Column Preparation Solution maximizes binding of the DNA to the membrane resulting in more consistent yields.
Note: Be sure to add ethanol to the Wash Solution Concentrate prior to first time use. See Preparation Instructions.
Note: When eluting with water, make sure that the pH of the water is between 5.5 and 8.5. Elution may also be performed using the Elution Solution diluted 10-fold with water.
A blood sample was isolated for genomic DNA using the GenElute Blood Genomic DNA Kit (NA2000). 10ng of genomic DNA was amplified using the GenomePlex WGA kit (WGA1) followed by purification using the GenElute PCR Clean Up Kit. SNP genotyping analysis was performed on non-amplified DNA and GenomePlex amplified DNA. GenomePlex WGA DNA genotyping results provided the same accuracy and quality of scores to non-amplified DNA.
1. Barker, D. L., et al. Two methods of whole-gen ome amplification enable accurate genotyping across a 2320-SNP linkage panel. Genomic Research, 14, 901-7 (2004).
2. Gribble, S., et al. Chromosome paints from single copies of chromosomes. Chromosome Research, 12, 143-51 (2004).
3. Thorstenson, Y. R., et al. An Automated Hydrodynamic Process for Controlled, Unbiased DNA Shearing. Genome Research, 8, 848-855 (1998).
To learn more about GenomePlex WGA technology visit: www.sigmaaldrich.com
GenomePlex is a registered trademark of Rubicon Genomics, Inc.
GenomePlex WGA technology patent pending.
GenElute and Jumpstart are trademarks of Sigma-Aldrich, Inc.
Nanodrop is a registered trademark of Nanodrop Technologies, Inc.
PicoGreen is a trademark of Molecular Probes, Inc.
BeadArray is a Trademark of Illumina, Inc.
TaqMan is a registered trademark of Roche Molecular Systems
Taq antibody licensed for in vitro research use under U.S. Patent No. 5,338,671 and 5,587,287, and corresponding patents in other countries.
This product is sold under license from Roche Molecular Systems, Inc. and Applied Biosystems.