Whole genome amplification for CGH analysis: Linker-adapter PCR as the method of choice for difficult and limited samples.

Division of Applied & Experimental Oncology, Institute of Cancer Research, Vienna University, Vienna, Austria.
Cytometry Part A (Impact Factor: 3.71). 10/2004; 61(1):26-34. DOI: 10.1002/cyto.a.20060
Source: PubMed

ABSTRACT Comparative genomic hybridization (CGH) is a powerful method to investigate chromosomal imbalances in tumor cells. However, DNA quantity and quality can be limiting factors for successful CGH analysis. The aim of this study was to investigate the applicability of degenerate oligonucleotide-primed PCR (DOP-PCR) and a recently developed linker-adapter-mediated PCR (LA-PCR) for whole genome amplification for use in CGH, especially for difficult source material.
We comparatively analyzed DNA of variable quality derived from different cell/tissue types. Additionally, dilution experiments down to the DNA content of a single cell were performed. FISH and/or classical cytogenetic analyses were used as controls.
In the case of high quality DNA samples, both methods were equally suitable for CGH. When analyzing very small amounts of these DNA samples (equivalent to one or a few human diploid cells), DOP-PCR-CGH, but not LA-PCR-CGH, frequently produced false-positive signals (e.g., gains in 1p and 16p, and losses in chromosome 4q). In case of formalin-fixed paraffin-embedded tissues, success rates by LA-PCR-CGH were significantly higher as compared to DOP-PCR-CGH. DNA of minor quality frequently could be analyzed correctly by LA-PCR-CGH, but was prone to give false-positive and/or false-negative results by DOP-PCR-CGH.
LA-PCR is superior to DOP-PCR for amplification of DNA for CGH analysis, especially in the case of very limited or partly degraded source material.

1 Bookmark
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Das Harnblasenkarzinom ist der zweithäufigste bösartige urologische Tumor. Der Grossteil (80%) der Tumore ist bei Erstdiagnose oberflächlich begrenzt, und weist eine hohe Rezidivrate auf. Die spezifischen ersten genetischen Veränderungen der Tumorgenese des Harnblasenkarzinoms, die nicht letal und eventuell einen Wachstumsvorteil beherbergen, sind bisher nicht bekannt. Ein Screening von Tumorvorstadien nach spezifischen Veränderungen auf chromosomaler Ebene in proliferierenden Zellen ermöglicht der gleichzeitige Einsatz zweier Methoden: der Interphase-Fluoreszenz-in-situ-Hybridisierung und der Immunhistochemischen(IHC)-Detektion des Proliferationsmarkers Ki67. Eine Zusammenstellung spezifischer Chromosomensonden (Urovysion; Abbott/Vysis) für diese Tumorentität wird bereits in der Routine-Diagnostik genutzt, um Rezidive und Tumor-Neuerkrankungen im Urin bzw. in Spülzytologien von Harnblasenkarzinom-Patienten nachzuweisen. In dieser Arbeit wurden 40 Harnblasengewebs-Biopsien untersucht, darunter waren 13 Hyperplasien, 12 Dysplasien, 7 CIS, 3 pTaG1 und 4 pT1G3-Fälle. Bei den Tumorvorstadien wurde darauf geachtet, dass es sich dabei möglichst um eine Erstdiagnose handelte. Zusätzlich wurde auf Einzelzell-Niveau mit Hilfe der Vergleichenden Genomischen Hybridisierung (CGH, Comparative Genomic Hybridisierung) das Genom nach Deletionen und Amplifikationen untersucht. Ziel war es, durch Kombination der drei Methoden (FISH, IHC und CGH) erste nicht letale typische chromosomale Aberrationen zu detektieren. Die Tumorvorstadien zeigten typische Chromosomenveränderungen, sowohl in den proliferienden Zellen, als auch in den umgebenden nicht-proliferierenden. Hyperplasien zeigten nur in nicht-proliferierenden Zellen Aberrationen. In proliferierenden Zellen von Dysplasien traten Verluste an den Chromosomen 9 und 13q auf, sowie Gewinne an den Chromosomen 9p, 12p, 17q, 18p, 22, X und Y. Involvierte Kandidatengene sind zahlreich und umfassen jene die Zellproliferation, Differenzierung, Apoptose und Stoffwechsel regulieren. In den Untersuchungen konnten chromosomale Veränderungen erstmalig in proliferierenden Zellen von Präkanzerosen detektiert werden, somit müssen diese Aberrationen mit einem proliferativen Vorteil und einer biologischen Relevanz einhergehen. Diese Arbeit wurde unterstützt durch die Deutsche Forschungsgemeinschaft (DFG-Nr.: Kn263/9-2). Bladder cancer is the second common malignant urological neoplasia. Most of the tumors are superficial (80%) at first diagnosis and reccur frequently. In order to understand the initial genetic aberrations reflecting growth advantage in bladder cancer we investigated first chromosomal aberrations and validated their biological potential at single cell level. Using multi-colour fluorescence in situ hybridisation (FISH; Urovysion) and Ki-67 immunohistochemistry first data was aquired and completed by laser-microdissecting single cells for single-cell comparative genomic hybridisation (CGH) analy-ses. Double staining of fluorescence in situ hybridisation (Urovysion, Vysis/Abbott) and Ki-67 immunohistochemistry was carried out on frozen tissue sections from 25/40 patients with pre-cancerous lesions of the bladder (13 hyperplasia, 12 dysplasia; those with preferably primary diagnosis; and further specimen from 7 carcinoma in situ, 3 pTaG1, 4 pT1G3). In addition 55 single cells of these precancerous lesions were laser-microdissected and analysed with single cell comparative genomic hybridisation (CGH). Focussing on the proliferating cells versus their non-proliferative neighbourhood in precan-cerous lesions of the bladder, chromosomal aberrations were detected in both types of cells. Proliferating hyperplastic cells showed almost a normal, diploid FISH and no further loss of chromosomal loci in the CGH-analysis. The CGH data of dysplasia cells showed mainly a loss of the chromosomal region 9p21 in proliferating cells, like expected from FISH results. Other chromosomal aberrations, depicted in dysplasia cells, were deletion of chromosome 9 and 13q as well as amplifications of the chromosomes 9p, 12p, 17q, 18p, 22, X and Y. In this regions many candidate genes, involved in regulation of cell proliferation and differentiation, apoptosis and metabolism, are located. These methods established are apt to show that genetic aberrations detected in early bladder lesions or normal urothelium are biologically relevant since found in proliferating cells. This work has been supported by the German Science Foundation (DFG, grant no: Kn263/9-2).
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Whole genome amplification (WGA) is usually needed in the genetic analysis of samples containing a low number of cells. In genome-wide analysis of DNA copy numbers by array comparative genomic hybridization (array-CGH) it is very important that the genome is evenly represented throughout the amplified product. All currently available WGA techniques are generating some degree of bias. A way to compensate for this is using a reference sample which is similarly amplified, as the introduced amplification bias will be leveled out. Additionally, direct labeling of the amplified DNA is performed to bypass the currently widely applied random primed labeling, which involves an additional amplification of the product and is introducing extra bias. In this article it is shown that equal processing of the test and reference sample is indeed crucial to generate an optimal array-CGH profile of amplified DNA samples. Also presented here is that the labeling method may significantly effect the array-CGH result, it is shown that with direct chemical labeling using platinum derivates (ULS labeling) optimal array-CGH results are obtained. We show that an optimized WGA strategy for both test and reference sample in combination with direct chemical labeling results in a reliable array-CGH profile of samples as low as a 30 cell equivalent.
    Cytometry Part A 09/2007; 71(8):585-91. · 3.71 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The availability of DNA samples that are of adequate quality and quantity is essential for any genetic analysis. The fields of forensic biology and clinical diagnostic pathology testing often suffer from limited samples that yield insufficient DNA material to allow extensive analysis. This study examined the utility of a recently introduced whole genome amplification method termed Multiple Displacement Amplification (MDA) for amplifying a variety of limited sample types that are commonly encountered in the fields of forensic biology and clinical diagnostics. The MDA reaction, which employs the highly processive bacteriophage φ29 DNA polymerase, was found to generate high molecular weight template DNA suitable for a variety of downstream applications from low copy number DNA samples down to the single genome level. MDA of single cells yielded sufficient DNA for up to 20,000,000 PCR assays, allowing further confirmatory testing on samples of limited quantities or the archiving of precious DNA material for future work. The amplification of degraded DNA material using MDA identified a requirement for samples of sufficient quality to allow successful synthesis of product DNA templates. Furthermore, the utility of MDA products in comparative genomic hybridisation (CGH) assays identified the presence of amplification bias. However, this bias was overcome by introducing a novel modification to the MDA protocol. Future directions for this work include investigations into the utility of MDA products in short tandem repeat (STR) assays for human identifications and application of the modified MDA protocol for testing of single cell samples for genetic abnormalities.


1 Download