Combined molecular genetic and cytogenetic analysis from single cells after isothermal whole-genome amplification.

Abstract

BACKGROUND:

Analysis of chromosomal aberrations or single-gene disorders from rare fetal cells circulating in the blood of pregnant women requires verification of the cells' genomic identity. We have developed a method enabling multiple analyses at the single-cell level that combines verification of the genomic identity of microchimeric cells with molecular genetic and cytogenetic diagnosis.

METHODS:

We used a model system of peripheral blood mononuclear cells spiked with a colon adenocarcinoma cell line and immunofluorescence staining for cytokeratin in combination with DNA staining with the nuclear dye TO-PRO-3 in a preliminary study to define candidate microchimeric (tumor) cells in Cytospin preparations. After laser microdissection, we performed low-volume on-chip isothermal whole-genome amplification (iWGA) of single and pooled cells.

RESULTS:

DNA fingerprint analysis of iWGA aliquots permitted successful identification of all analyzed candidate microchimeric cell preparations (6 samples of pooled cells, 7 samples of single cells). Sequencing of 3 single-nucleotide polymorphisms was successful at the single-cell level for 20 of 32 allelic loci. Metaphase comparative genomic hybridization (mCGH) with iWGA products of single cells showed the gains and losses known to be present in the genomic DNA of the target cells.

CONCLUSIONS:

This method may be instrumental in cell-based noninvasive prenatal diagnosis. Furthermore, the possibility to perform mCGH with amplified DNA from single cells offers a perspective for the analysis of nonmicrochimeric rare cells exhibiting genomic alterations, such as circulating tumor cells.

• Authors: Thomas Kroneis, Jochen B Geigl, Amin El-Heliebi, Martina Auer, Peter Ulz, Thomas Schwarzbraun, Gottfried Dohr, Peter Sedlmayr
• Journal: Clinical chemistry (Clin Chem) Vol. 57 Issue 7 Pg. 1032-41 (Jul 2011) ISSN: 1530-8561 [Electronic] England
• PMID: 21558453 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Keratins

Topics

• Chimerism
• Chromosome Aberrations
• Comparative Genomic Hybridization (methods)
• Cytogenetic Analysis (methods)
• DNA Fingerprinting (methods)
• Female
• Fluorescent Antibody Technique
• Genome, Human
• HT29 Cells
• Humans
• Keratins (metabolism)
• Leukocytes, Mononuclear (cytology, metabolism)
• Metaphase
• Neoplastic Cells, Circulating (metabolism, pathology)
• Nucleic Acid Amplification Techniques
• Pregnancy
• Prenatal Diagnosis (methods)
• Sequence Analysis, DNA (methods)
• Single-Cell Analysis (methods)