GenomePlex® Complete Whole Genome Amplification Kit (WGA2) - Protocol

Product Description

GenomePlex is a Whole Genome Amplification (WGA) method that allows the researcher to generate a representative, ~500-fold amplification of genomic DNA. 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 library is then PCR amplified using universal oligonucleotide primers and a limited number of cycles.

The GenomePlex Complete WGA Kit contains an optimized enzyme that decreases the background in the reaction. The WGA DNA Polymerase successfully produces a negative control sample without product. This result provides more accuracy in downstream PCR or hybridization techniques.

WGA has been used in a variety of applications(see Ref) and is suitable for use with purified genomic DNA from a variety of sources including blood card, whole blood, formalin fixed tissues, and cultured cells. GenomePlex Complete Whole Genome amplification kit requires a minimum 10 ng of starting DNA, which after PCR can yield >10 µg of WGA product. After purification, the WGA product can be analyzed in a manner similar to any genomic or chromosomal DNA sample. A number of downstream applications may be performed including TaqMan® assays, microsatellite analysis, SNP analysis, sequencing, etc.

Reagents Provided

  10 RXN 50 RXN 500 RXN
10x Fragmentation Buffer Catalog Number F4304 12 µL 55 µL 550 µL
1x Library Preparation Buffer Catalog Number L7167 22 µL 110 µL 1.1 ml
Library Stabilization Solution Catalog Number L7292 12 µL 55 µL 550 µL
Library Preparation Enzyme Catalog Number E0531 12 µL 55 µL 550 µL
10x Amplification Master Mix Catalog Number A5604 80 µL 410 µL 4.1 ml
WGA DNA Polymerase Catalog Number W3891 55 µL 275 µL 2.75 ml
Water, Molecular Biology Reagent Catalog Number W4502 1 x 1.5 ml 2 x 1.5 ml 2 x 20 ml
Control Human Genomic DNA Catalog Number D7192 10 µL (5 ng/µL) 10 µL (5 ng/µL) 50 µL (5 ng/µL)

Storage/Stability

All components should be stored at –20 °C. When thawed for use, components should be kept on ice. Stability of the Library Preparation Enzyme will be affected if stored warmer than –20 °C or allowed to remain for long periods at temperatures over 4 °C.

Procedure

The WGA process is divided into fragmentation, OmniPlex library generation, and PCR amplification. The first two steps, fragmentation and library generation, should be carried out without interruption, as the ends of the library DNA can degrade thus affecting subsequent steps. OmniPlex library DNA, generated in the stepped isothermal reactions, can be stored up to three days at –20 °C without detectable differences to the process. The final WGA DNA should be stored at –20 °C and is as stable as any comparably stored genomic DNA sample.

The starting amount of DNA is critical. When using a complex starting material, such as human genomic DNA, the gene bias in the resulting Complete WGA product is significantly altered if the quantity of input DNA is reduced. One ng of human genomic DNA affords product with gene representation that varies 2 to 10-fold from the original material, while product yield is only ~50% lower. Less complex genomes such as bacterial DNA can give good representation with as little as 1 ng of input DNA.

GenomePlex can be used on archival fixed tissue DNA or degraded samples provided that the extracted DNA is 200 bp or greater in size. However, greater quantities of damaged DNA are required to afford acceptable yields of final product. We recommend using up to 100ng of fixed tissue DNA and following the complete protocol, including fragmentation.

Experiments should be performed along with a positive control DNA sample, such as the Control Human Genomic DNA included in the kit.

Fragmentation

1. Isolate DNA sample and quantify concentration by UV absorption (260 nm). Prepare DNA solution of 1ng/mL

2. Add 1 µL of 10x Fragmentation Buffer to 10 µL of DNA (1 ng/µL) sample in a PCR tube or multiwell strip/plate.

3. Place the tube/plate in a thermal block or cycler at 95 °C for EXACTLY 4 minutes. Note, the incubation is very time sensitive. Any deviation may alter results.

4. Immediately cool the sample on ice, then centrifuge briefly to consolidate the contents.

Library Preparation

5. Add 2 µL of 1x Library Preparation Buffer to each sample.

6. Add 1 µL of Library Stabilization Solution.

7. Vortex thoroughly, consolidate by centrifugation, and place in thermal cycler at 95 °C for 2 minutes.

8. Cool the sample on ice, consolidate the sample by centrifugation, and return to ice.

9. Add 1 µL of Library Preparation Enzyme, vortex thoroughly, and centrifuge briefly.

10. Place sample in a thermal cycler and incubate as follows:
    16 °C for 20 minutes
    24 °C for 20 minutes
    37 °C for 20 minutes
    75 °C for 5 minutes
    4 °C hold

11. Remove samples from thermal cycler and centrifuge briefly. Samples may be amplified immediately or stored at –20 °C for three days.

Amplification

12. A master mix may be prepared by adding the following reagents to the 15 µL reaction from step 11:

    7.5 µL of 10X Amplification Master Mix
    47.5 µL of Water, Molecular Biology Reagent
    5 µL of WGA DNA Polymerase

13. Vortex thoroughly, centrifuge briefly, and begin thermocycling. The following profile has been optimized for a PE 9700 or equivalent thermocycler:
    Initial Denaturation    95 °C for 3 minutes
    Perform 14 cycles as follows:
    Denature                94 °C for 15 seconds
    Anneal/Extend        65 °C for 5 minutes

After cycling is complete, maintain the reactions at 4 °C or store at –20 °C until ready for analysis or purification. The stability of WGA DNA is equivalent to genomic DNA stored under the same conditions.

WGA2

Purification of the final product is recommended before use in subsequent applications. Complete WGA amplified DNA may be purified with Sigma PCR Cleanup Kit (Catalog Number NA1020) or standard purification methods that isolate single and double stranded DNA. Once purified, the DNA can be quantified by measuring absorbance, assuming that one A260 unit is equivalent to 50 ng/µL DNA. Measurement techniques such as PicoGreen® will often underestimate the actual WGA DNA yield, since single stranded DNA might be generated during amplification.

Product Profile

A 10 ng sample of standard human genomic DNA must yield 3–7 µg of product. A no template control must afford no or little yield by the gel. The quality and representation of amplification is determined by real-time PCR using primer sets for eight separate loci.

References

  1. Massively parallel sequencing reveals the complex structure of an irradiated human chromosome on a mouse background in the Tc1 model of Down syndrome
  2. Nanobody-based chromatin immunoprecipitation
  3. Methylome-wide comparison of human genomic DNA extracted from whole blood and from EBV-transformed lymphocyte cell lines
  4. X chromosome gene methylation in peripheral lymphocytes from monozygotic twins discordant for scleroderma.
  5. Three murine leukemia virus integration regions within 100 kilobases upstream of c-myb are proximal to the 5' regulatory region of the gene through DNA looping.
  6. Gene expression and epigenetic changes by furan in rat liver.
  7. Chromosomal copy number alterations are associated with persistent lymph node metastasis after chemoradiation in locally advanced rectal cancer.
  8. A comparison of transcriptome and epigenetic status between closely related species in the genus Arabidopsis.
  9. Unexpected results in the constitution of small supernumerary marker chromosomes.
  10. Defining NOTCH3 target genes in ovarian cancer
  11. Transcriptional control of cell cycle-dependent kinase 4 by Smad proteins--implications for Alzheimer's disease
  12. Barker, D. L., et al., Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel. Genome Res., 14, 901-907 (2004).
  13. Gribble, S., et al., Chromosome paints from single copies of chromosomes. Chromosome Res., 12, 143-151 (2004).
  14. Thorstenson, Y. R., et al., An Automated Hydrodynamic Process for Controlled, Unbiased DNA Shearing. Genome Res., 8, 848-855 (1998).

 

Troubleshooting Guide

Observation Cause Recommended Solution
Low yield after cycling Sample contains PCR inhibitors or high buffer salts. Dialysis in a suitable microdialysis unit may dilute the inhibiting components. Loss of DNA may occur in this process, so quantitation of the dialyzed product is highly recommended.
Input DNA is severely degraded or was less than 10 ng. Amplification of insufficient DNA quantities often results in poor yield or poor representation in the final product. Some templates can be rendered amplifiable by using more input DNA. Successful WGA amplification has been performed with degraded samples by increasing starting template to 25–100 ng.
More enzyme is required. WGA yield suffers when limiting amounts of DNA polymerase are used. We recommend a minimum of 5 µL of WGA DNA Polymerase per 75 µL reaction. This is preferable to adding cycles as the resulting DNA may suffer from amplification bias.
Post reaction purification was inappropriate. We recommend Sigma’s PCR Cleanup Kit (NA1020). The method must retain single and double stranded DNA.
Fragmentation reaction is too long or short. The four-minute fragmentation time was found to give optimal results over a wide variety of DNA samples. Too little or no fragmentation will afford low yields and poor gene representation in the resulting WGA product. A ten-minute fragmentation step will also give low yields in almost all cases, because a significant fraction of the DNA is now too small to allow efficient library production.
qPCR shows significant bias in WGA representation for my gene of interest. Inappropriate controls Genomic DNA can only be compared to GenomePlex WGA once the control DNA has been sheared. We recommend using several pooled samples that have been subjected to the fragmentation protocol (steps 1–4) or compare against DNA subjected to hydroshearing.3
DNA sample is limited or degraded. See low yield comments.
Negative (no template) control affords product. Reagents have been contaminated by an outside source. One or more reagents have been contaminated with DNA.  While this may not affect your results, a clean no template control can be re-achieved only by replacing the affected component.

Frequently Asked Questions

1. How does GenomePlex work? Genomic DNA is randomly fragmented and the resulting product is manipulated to attach a common sequence at each DNA end. This library of fragments is amplified using fourteen rounds of PCR.

2. What if fragmentation is allowed to proceed for more or less than four minutes? The 4-minute fragmentation time was found to give optimal results over a wide variety of DNA samples. Too little or no fragmentation will afford low yields and poor gene representation in the resulting WGA product. A ten minute fragmentation step will also give poor WGA yields in almost all cases, because a significant fraction of the DNA is now too small to allow efficient library production.

3. What is the average size of fragmented DNA? The mean size after the fragmentation step is ~0.4kb.

4. Can GenomePlex be used on archival fixed tissue DNA or degraded samples? Yes, provided that the extracted DNA is 200 bp or greater in size. However, more damaged DNA is required to afford acceptable yields of final product. We recommend using up to 100 ng of fixed tissue DNA and following the complete protocol, including fragmentation. See the troubleshooting guide under low yield, input DNA is degraded.

5. Can I use less input DNA in the GenomePlex protocol? When using a complex starting material, such as human genomic DNA, the gene bias in the resulting GenomePlex output is significantly altered if the input DNA is reduced. An input of 1 ng of human genomic DNA affords product with gene representation that varies 2 to 10-fold over the original material, even though the yield is only ~50% lower. However, less complex genomes such as bacterial DNA can give good representation with only 1 ng of input DNA.

6. How should WGA DNA be purified? Is there a preferred way to quantify GenomePlex DNA? We recommend purifying GenomePlex DNA using Sigma’s PCR Cleanup Kit (Catalog Number NA1020) before it is used in any downstream process. Once purified, the DNA can be quantified by measuring absorbance, assuming that one A260 unit is equivalent to 50 ng/µL DNA. Measurement techniques such as PicoGreen will often underestimate the actual WGA DNA yield, since single stranded DNA might be generated during amplification.

7. How can I store GenomePlex DNA? Where can I stop during the GenomePlex process? The WGA process can be divided into fragmentation, generation of the OmniPlex library, and PCR amplification. Fragmented DNA should be processed immediately, as the ends of this DNA can degrade and will affect subsequent steps. OmniPlex library DNA, generated in the stepped isothermal reactions, can be stored up to three days at –20 °C without any detectable differences to the process. The final WGA DNA should be stored at –20 °C and is as stable as any comparably stored genomic DNA sample.
8. What studies were conducted to determine sequence fidelity for this whole genome amplification method? The GenomePlex method was tested for representation during development of the product by using 107 different human primer sets along with PCR and quantitative PCR. The sets are all from the National Center for Biotechnology UniSTS database.* While this subset of 107 DNA sequences represents a small fraction of the
20–30,000 human genes, it is a good statistical representation of the human genome.

Collaborators have also published work showing the representation of the amplification obtained using GenomePlex.1,2

Disclaimer

This product is for research use only. Use of this product for human or animal therapeutic or diagnostic purposes requires licensing from Rubicon Genomics, Inc. Unauthorized use violates proprietary rights of Rubicon Genomics, Inc (www.rubicongenomics.com).

NOTICE TO PURCHASER: LIMITED LICENSE

Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US:  5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to expired US Patent No. 5,079,352.  The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim, no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. 

GenomePlex and OmniPlex are registered trademarks of Rubicon Genomics.
PicoGreen is a registered trademark of Molecular Probes, Inc
TaqMan is a registered trademark of Roche Molecular Systems

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