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Whole genome amplification from dried blood spots using illustra™ Ready-To-Go™ GenomiPhi™ V3

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Fig 1. illustra Ready-To-Go GenomiPhi V3 DNA Amplification Kit and an FTA card spotted with human whole blood.

Whatman FTA cards are cellulose-based sample collection tools that simplify the collection, shipment, storage, and purification of nucleic acids from a wide variety of sources. Human, animal, plant, or microbe samples are applied directly onto the card. Chemicals in the FTA card lyse the cells, denature proteins, and immobilize the nucleic acids.  After drying, DNA is protected from enzymatic, microbial, oxidative, and free-radical damage. The samples can be stored for years at room temperature before analysis (more than 17 years for blood on FTA cards). For analysis or use of DNA stored on FTA cards, a small disc is cut from the sample area. For typical blood samples (assuming 5000 white blood cells per μl of whole blood), a 1.2 mm disc from a dried blood spot may contain approximately 5 ng of DNA, and a 3 mm disc approximately 30 ng.

The amount of genomic DNA that is obtained from the cards can sometimes be limiting when larger quantities are required for complete genomic analyses. This article describes a simple method for whole genome amplification from FTA blood cards with minimal handling using the Ready-To-Go GenomiPhi V3 DNA Amplification Kit (GE Healthcare). The method produces microgram quantities of high molecular weight DNA that can be used directly in downstream genomics applications.

Here we describe a simple method for elution of DNA from FTA dried blood spot samples in a form that is suitable for whole genome amplification using the illustra Ready-To-Go GenomiPhi V3 DNA Amplification Kit. This method consistently yielded over 15 micrograms of high molecular weight DNA from individual 1.2 mm or 3 mm punched discs. STR profiling showed full concordance between amplified and unamplified DNA.

Introduction

The illustra Ready-To-Go GenomiPhi V3 DNA Amplification Kit from GE Healthcare Life Sciences provides an ambient temperature-stable reaction mix for whole genome amplification (WGA). Using Phi29 DNA polymerase and random hexamer primers, isothermal WGA is achieved by a multiple strand displacement amplification (MDA) mechanism (1), producing microgram quantities of high molecular weight copies from nanogram amounts of genomic DNA (gDNA). The proofreading 3’–5’ exonuclease activity of Phi29 DNA polymerase ensures high fidelity amplification (2, 3). MDA provides excellent genome coverage and is a well-established method used successfully in many genomics applications (4, 5, 6).

Whatman FTA cards are cellulose-based sample collection tools that simplify the collection, shipment, storage, and purification of nucleic acids from a wide variety of sources. Human, animal, plant, or microbe samples are applied directly onto the card. Chemicals in the FTA card lyse the cells, denature proteins, and immobilize the nucleic acids.

After drying, DNA is protected from enzymatic, microbial, oxidative, and free-radical damage. The samples can be stored for years at room temperature before analysis (more than 17 years for blood on FTA cards).

For analysis or use of DNA stored on FTA cards, a small disc is cut from the sample area. For typical blood samples (assuming 5000 white blood cells per µl of whole blood), a 1.2 mm disc from a dried blood spot may contain approximately 5 ng of DNA, and a 3 mm disc approximately 30 ng. Therefore, the amount of genomic DNA that is obtained from the cards can be limiting, and additional larger samples are often required for complete genomic analyses. This application note describes a simple method for WGA from FTA blood cards with minimal handling, using the Ready-To-Go GenomiPhi V3 DNA Amplification Kit. The method produces microgram quantities of high molecular weight DNA that can be used directly in downstream genomics applications.

 

Methods

Whole genome amplification from FTA dried blood spots

To prepare dried blood spots, 60 µl of human whole blood was spotted onto an FTA Classic Card (GE Healthcare; WB120205), which was allowed to dry overnight at room temperature and stored desiccated until required. Both 1.2 and 3 mm discs were punched from the FTA dried blood spot using sterile Harris Micro punches with a cutting mat. Triplicate punches for each diameter were processed and subjected to whole genome amplification as described in the following standard protocol. Control WGA reactions were carried out using genomic DNA purified from blood with the illustra blood genomicPrep Mini Spin Kit (GE Healthcare; 28-9042-64). Amplification products were quantified using Quant-iT™ PicoGreen™ dsDNA Reagent (Life Technologies; P7589).

 

Standard protocol

A. Punch processing (alkaline pH elution)

  1. From the dried blood spot region of the FTA card, punch out 1.2 or 3 mm diameter discs.
  2. Place each disc in a microcentrifuge tube and add FTA Purification Reagent (GE Healthcare; WB120204; 200 µl for 1.2 mm and 500 µl for 3 mm discs). Mix by pipetting up and down several times (do not vortex). Incubate for 5 min at room temperature. Carefully remove the liquid using a pipette. Repeat 2 times for a total of 3 washes.
  3. Add TE buffer (pH 8.0; 200 µl for 1.2 mm and 500 µl for 3 mm discs) to each tube. Mix by pipetting up and down several times (do not vortex). Incubate for 5 min at room temperature. Carefully remove the liquid using a pipette. Repeat the wash. Remove as much liquid as possible at the end of the second wash.
  4. Add 10 µl of cell lysis/denaturation solution (400 mM KOH, 5 mM EDTA) to each damp disc and mix well with gentle tapping.
  5. Incubate the samples on ice for 10 min.
  6. Add 20 µl of neutralization buffer (300 mM Tris-HCl, 200 mM HCl; prepared by mixing 3 ml of 1 M Tris-HCl [pH 7.5] and 2 ml of 1 M HCl with 5 ml of water) to each cell lysate, mix well with gentle tapping, and store samples on ice.
  7. Add 2 µl of each neutralized lysate to 8 µl of PCR-grade water and mix by pipetting up and down several times or by gently tapping to create the final amplification template concentration.

 

B. Whole genome amplification using the Ready-To-Go GenomiPhi V3 Kit

  1. To the 10 µl of amplification template, add 10 µl of the 2x denaturing buffer from the Ready-To-Go GenomiPhi V3 DNA Amplification Kit (25-6601-24, -96, or -97), and pipette the whole 20 µl onto a Ready-To-Go GenomiPhi V3 cake. Mix by gently pipetting up and down several times. Keep all amplification reactions on ice prior to incubation at 30°C.
  2. Incubate the samples at 30°C for 2 h.
  3. Heat the samples to 65°C for 10 min then cool to 4°C. Heating is required to inactivate the exonuclease activity of the DNA polymerase, which would otherwise degrade the amplification product.
  4. Store amplified DNA at -20°C. Ready-To-Go GenomiPhi V3 amplification products should be stored and treated as genomic DNA. Minimize freeze-thaw cycles.

 

Agarose gel analysis

Samples of each WGA reaction were digested with EcoRI restriction endonuclease and run on a 1% agarose gel alongside the corresponding undigested WGA reaction to determine whether the amplified DNA was restriction endonuclease sensitive. For each digestion reaction, 10 µl of amplified product was incubated with 10 units of enzyme overnight at 37°C. A positive WGA control, which was amplified from DNA purified using the illustra blood genomicPrep Mini Spin Kit, was included together with a ‘no template’ control.

 

STR genotyping

To assess the quality of amplified DNA, we performed STR genotyping analysis. All materials in this section except the Ready-To-Go Kit are from Life Technologies. DNA was eluted from 3 mm discs and amplified with Ready-To-Go GenomiPhi V3 as described above. STR profiles were obtained from 2 ng

of amplified DNA or 2 ng of unamplified control DNA using the AmpFlSTR™ Identifiler™ PCR Amplification Kit (4322288) according to the manufacturer’s protocol. STR reactions were performed on a GeneAmp™ PCR System 9700. Capillary electrophoresis was conducted on a 3130xL Genetic Analyzer followed by analysis using GeneMapper™ ID v3.2 Software.

 

Results and discussion

 

Yield and size distribution of genomic DNA amplified from FTA dried blood spots

Yields of DNA after amplification with Ready-To-Go GenomiPhi V3 were consistently between 15 and 18 µg (Table 1) for both 1.2 and 3 mm punched discs. These yields are within the Kit’s specification range of 12–20 µg.

Table 1. Yields of DNA amplified from discs punched from FTA dried blood spots were measured by Quant-iT PicoGreen dsDNA assay

Disc Size                                                             3 mm   3 mm   3 mm   1.2 mm  1.2 mm   1.2 mm

Replicate #                                                           1           2           3              1              2              3

Yield of GenomiPhi amplified DNA (μg)       17.4     15.6      17.4         15.7        16.9        16.0 

Agarose gel analysis in Figure 2 shows that undigested WGA product from FTA dried blood spots is of high molecular weight (> 10 kb), as is typical of GenomiPhi reactions. In samples digested with EcoRI, high molecular weight DNA transitions to a lower molecular weight smear as expected from amplified genomic DNA. No DNA was visible for the ‘no template’ control (data not shown).

 

Table 2. Allele calls for 16 loci determined by STR profiling

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Fig 2. Agarose gel analysis (1%): odd lanes contain undigested DNA; even lanes contain DNA digested with EcoRI. Lanes 1–6, 1.2 mm punch; lanes 7–12, 3 mm punch; lanes 13–16, positive control. M is a molecular weight marker.

 

STR analysis of genomic DNA amplified from FTA dried blood spots

DNA was eluted from FTA dried blood spots according to the alkaline denaturation protocol described here, amplified with Ready-To-Go GenomiPhi V3, and subjected to STR amplification. STR analysis produced the expected allele calls for the full profile of 15 alleles (plus amelogenin) for all replicates tested (Fig 3, Table 2). Peak heights are measured in relative fluorescence units (RFU).

 

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Fig 3. Output from GeneMapper software showing the 15 AmpFLSTR Identifiler  alleles (+ amelogenin) for GenomiPhi V3 WGA from FTA eluted DNA.

 

Conclusions

 

Storage of blood and biological samples on FTA cards offers outstanding convenience and efficiency with simple handling and ambient temperature storage. Key to this utility is the ability to effectively elute DNA of sufficient quality for subsequent use. Typically, sample is recovered by treatment of small punched discs, which are washed to remove impregnated chemicals prior to DNA elution. Smaller punched discs are preferable because they leave the remainder of the dried blood spot for future use; however, the amount of DNA present on the disc can be limiting for downstream assay requirements.

We have demonstrated that the illustra Ready-To-Go GenomiPhi V3 DNA Amplification Kit can be effectively combined with sample collection on FTA cards to generate microgram quantities of high molecular weight DNA from 1.2 and 3 mm punched discs. A simple alkaline elution protocol has been described that facilitates elution of DNA from FTA dried blood spots in a form that is suitable for whole genome amplification. Subsequent STR analysis of DNA amplified from FTA discs provided full profiles with all alleles correctly identified.

The protocol described here helps to preserve valuable samples and enables researchers to generate sufficient high-quality DNA from those samples for multiple downstream applications. Furthermore, we provide an entirely ambient temperature-stable solution from sample collection to whole genome amplification, facilitating logistics and study design particularly in remote areas or settings with limited infrastructure.

In summary:

  • We have presented a method by which DNA can easily be recovered from FTA dried blood spots using alkaline pH elution.
  • DNA eluted from FTA dried blood spots using this method is suitable for whole genome amplification with the illustra Ready-To-Go GenomiPhi V3 DNA Amplification Kit. We generated DNA yields in excess of 15 µg from both 1.2 and 3 mm punches.
  • Amplified DNA generated with GenomiPhi V3 following alkaline pH elution is suitable for subsequent genetic analyses including STR profiling, which showed full concordance between amplified and unamplified DNA.

 

References

 

  1. Dean, F. B. et al. Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099 (2001).
  2. Esteban, J. A. et al. Fidelity of phi 29 DNA polymerase: comparison between protein-primed initiation and DNA polymerization. J. Biol. Chem. 268, 2719–2726 (1993).
  3. Nelson, J. R. et al. TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing. BioTechniques 32, S44–S47 (2002).
  4. Reyes, A. Viruses in the faecal microbiota of monozygotic twins and their mothers. Nature 466,

334–338 (2010).

  1. Hughes, S. et al. The use of whole genome amplification to study chromosomal changes in prostate cancer: insights into genome-wide signature of preneoplasia associated with cancer progression. BMC Genomics 7, 65 (2006).
  2. Pask, R. et al. Investigating the utility of combining phi29 whole genome amplification and highly multiplexed single nucleotide polymorphism BeadArray genotyping. BMC Biotechnol. 4, 15 (2004).

 

 

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