Single Cell Whole Genome Amplification: Unleashing a World within a Cell

By: Chad T. Brueck, Deborah L. Vassar, Clyde Brown, Ernie Mueller, LSI Edition 23

Chad T. Brueck, Deborah L. Vassar, Clyde Brown, Ernie Mueller, Sigma-Aldrich Corp., St. Louis, MO, USA

Introduction

Analyzing genomic DNA at the level of the single cell will provide the ultimate level of variation analysis. Unfortunately, amplifying the picogram quantities of DNA in a single cell has been difficult. The recent advent of commercial kits for whole genome amplification has provided scientists the means to amplify the information from ~3000 cells, affording amplification that is a complete and faithful representation of the original DNA. While advancing the field of DNA variation analysis, these kits have utility in fields such as oncology, molecular pathology, and in vitro fertilization where analyzing the DNA from a single cell is the optimal choice. The GenomePlex Single Cell Whole Genome Amplification Kit was created to address these limitations.

GenomePlex is a whole genome amplification (WGA) method that allows representative amplification of genomic DNA. The kit utilizes a proprietary amplification technology based upon initial nicking of genomic DNA to relax the template for the subsequent hybridization of a unique dual-function primer. One function of the primer is to representatively anneal roughly every 500 bp throughout the entire genome. After annealing, the adjacent template is extended to create PCR amplifiable OmniPlex fragments flanked by universal priming sites, creating a library, which represents the entire genome. The OmniPlex library is then PCR amplified using universal oligonucleotide primers complementary to the initial primer sequence; hence, the second function of the WGA primer. The Single Cell WGA Kit has been optimized to amplify the genome of a single cell. WGA of a single cell results in a million-fold amplification yielding microgram quantities of gDNA. After purification, the Single Cell WGA product can be analyzed in a manner similar to any genomic or chromosomal DNA sample.

This article demonstrates the level of analysis available downstream of single cell WGA. The utility of Genomeplex Single Cell WGA Kit has been shown over a variety of single cell samples and numerous downstream applications within our own labs, and from customers using our product.

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Results Generated with Single Cell WGA

Single Cell WGA was performed on tobacco protoplasts isolated from single cells (diluted to single cell concentrations), see Figure 1. Amplification resulted in approximately 6 μg per cell. The quality of amplified DNA was analyzed via SYBR® Green qPCR (see Figure 2). Relatively low allelic dropout (ADO) was observed. The amount of DNA accessible due to cell state and/or cell quality can effect the representation of downstream-amplified DNA. Typically, 25-50% of the genome is inaccessible to amplification at the single cell level; hence, downstream analyses are not as robust compared to amplification of nanogram-quantities of DNA. Further research has suggested that amplification of 5–10 cells is the minimum number of cells required to afford quality analytical resolution.


Figure 1. WGA Results


Figure 2. qPCR Results

To demonstrate the reproducibility of the GenomePlex Single Cell Kit, single human leukemia U937 cells were isolated using Flow Cytometric Analysis and Sorting (FACS), lysed and amplified via the Single Cell WGA Kit (see Figure 3). The DNA was then purified with the GenElute™ PCR Cleanup Kit. An estimated million-fold amplification from the WGA process resulted in a final yield ranging from 5.4–6.2 μg. In addition, WGA was performed on single human leukemia U937 cells isolated by laser capture microdissection (LCM), see Figure 4. As visualized on the agarose gel, yield and size are consistent from cell to cell. The Single Cell WGA Kit produces consistent yield and size range as visualized by a 1% agarose gel.


Figure 3. FACS Isolated


Figure 4. LCM Isolated

FACS isolated WGA DNA was then utilized in a MGB Eclipse™ SNP (Single Nucleotide Polymorphism) assay by quantitative PCR on the ABI 7700. The MGB Eclipse Probe System facilitates allelic discrimination due to its ability to allow post-PCR dissociation curves. Dissociation curves allow determination of the melting point (Tm). Different alleles will have a substantially different Tm value. The FAM and TET fluorescent signals help distinguish homozygous wild, heterozygous and homozygous mutant types. The WGA amplified single cell DNA was tested for 3 SNPs, MTHFR01, IGF1R-03 and NAT1-01. A positive, unamplified control was evaluated with each group. The data indicates that all of the single cells were the same allele for each SNP tested.

Dr. Michael Speicher (Medical University of Graz, Austria) demonstrated the utility of GenomePlex amplification when analyzing renal carcinoma at the resolution of a single cell. Two DNA samples of a known genotype were amplified using the GenomePlex® Single Cell WGA Kit. Each library of amplified DNA was labeled and then hybridized to normal human metaphase spreads. The results were compared to the known genotype for each sample. In the first sample, ten kidney tumor cells were subjected to the single cell protocol. A control was also performed using a single cell from a normal female. The expected results are shown as a diagram where regions of chromosome deletion or amplification are indicated by red or green lines, respectively. The genotyping data is shown as a black line and CGH representation is measured as underrepresented (below the red line) or overrepresented (above the green line). In all cases the WGA DNA had representation values identical to the expected, previously determined values.

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Conclusions

Analyzing the genomic material in a single cell has long been desirable but heretofore unachievable due to the miniscule amount of DNA available for analysis. The GenomePlex Single Cell Whole Genome Amplification Kit opens the door to the world within the single cell. This process amplifies the DNA from a single cell a million fold, allowing the genetic analysis of the ultimate biological unit and opening the secrets to maturation, regeneration and genetic diseases. GenomePlex is compatible with cells isolated by dilution protocols, flow sorting, and laser capture as shown in the data. This DNA can be analyzed by SNP analysis, gene expression studies and comparative genomic hybridization.

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Acknowledgements

We would like to thank Barbara Pilas from University of Illinois at Urbana-Champaign and Joy Eslick from Saint Louis University for their single cell FACS work. In addition, the University of Albany’s Center for Functional Genomics has kindly provided laser-captured microdissected cells to further validate the GenomePlex Single Cell WGA Kit.

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References

  1. Barker, DL, et al., (2004) Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel. Genome Research, Vol 14: 901-907
  2. Bergen A, et al., (2005) Comparison of Yield and Genotyping Performance of Multiple Displacement Amplification and OmniPlex (trade mark) Whole Genome Amplified DNA Generated from Multiple Sources. Human Mutation, Vol 26: 262-270
  3. Gribble, S, et al., (2004) Chromosome paints from single copies of chromosomes. Chromosome Research, Vol 12: 143-151
  4. Hughes S, Lasken R, (2005) Whole Genome Amplification. Scion: Bloxham
  5. Hughes S, et al., (2005) The Use of Whole Genome Amplification in the Study of Human Disease. Prog Biophys, Mol. Biol. 88, 173-189
  6. Kiechle F, et al., (2004) The –Omics Era and its Impact. Arch Pathol Lab Med, Vol 128: 1337-1345

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