BRIEF REPORT |
First Systematic CGH-based Analyses of Ancient DNA Samples of Malformed Fetuses Preserved in the Meckel Anatomical Collection in Halle/Saale (Germany)
Institut für Humangenetik, Chromosomendiagnostik und Molekuluare Zytogenetik, Charité, Campus Virchow Klinikum, Humboldt-Universität Berlin, Berlin, Germany (HT,AG), and Institut für Anatomie und Zellbiologie, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany (RK,RS,LG)
Correspondence to: H. Tönnies, Institut für Humangenetik, Campus Virchow Klinikum, Charité, Berlin, Augustenburger Platz 1, 13353, Berlin, Germany. E-mail: holger.toennies{at}charite.de
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Summary |
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Key Words: ancient DNA Meckel Anatomical Collection comparative genomic hybridization polymerase chain reaction
COMPARATIVE GENOMIC HYBRIDIZATION (CGH) is a well-proven molecular cytogenetic approach for the genome-wide analysis of chromosomal gains and losses in high-molecular-weight DNA probes without preparing chromosomes of the test sample (Kallioniemi et al. 1992). Using this molecular cytogenetic approach, the identification of chromosomal imbalances can be achieved with cytogenetic resolution in a single hybridization experiment (Tönnies et al. 2001
).
The term ancient DNA (aDNA) describes DNA that can be extracted mostly in small amounts and at different stages of degradation from non-living clinical, museal, archeological, and paleontological samples (Herrmann and Hummel 1993). The age of the source material can differ from a few years to thousands of years. In the literature, different molecular genetic investigations on aDNA are described (for review, see Marota and Rollo 2002
). We performed the first successful CGH analyses on aDNA extracted from a bronze-age human individual and a 262-year-preserved malformed fetus without former PCR amplification, as described previously (Tönnies et al. 1998
; Hummel et al. 1999
).
The aim of the Meckel Collection study is to gain deeper insights into the effect of different aDNA extraction and amplification protocols on the quality of CGH results. Additionally, we are interested in the possible identification of cytogenetic imbalances in clinically well-described "ancient" malformed fetuses and in further testing of different confirmatory allele-specific PCR analyses on genomic aDNA probes.
To date, we have extracted aDNA from 19 different ethanol- or formalin-fixed umbilical cord and muscle samples of malformed fetuses under sterile conditions. Recurrent DNA extractions have been performed using standard phenol-chloroform protocols and the Invisorb Forensic Kit as recommended by the manufacturer (Invisorb; Berlin, Germany). The Invisorb Forensic Kit gave repeatedly better results concerning PCR-amplifiable DNA in comparison to standard phenol-chloroform-based protocols. However, in all cases, the total quantity of aDNA extracted was below the quantity needed for successful CGH experiments. As shown in previous experiments, the total amount of genomic DNA needed for detecting known chromosomal imbalances by CGH on metaphase spreads must be 50 ng in 10 µl hybridization solution (5 ng/µl) for a 324-mm2 hybridization area (Hummel et al. 1999). In the literature, different whole-genome amplification strategies, mainly degenerate oligonucleotide primer (DOP)-PCRbased (Telenius et al. 1992
) strategies, are described for the amplification of aDNA probes (Kittler et al. 2002
). Testing different amplification protocols with aDNA extracted from ethanol-and unbuffered formalinfixed probes, a simple protocol based on using modified DOP-Primer (5'-CCG ACT GCA GNN NNN NAT GTG G-3') and the Expand High Fidelity PCR System (HIFI-DOP) (Roche, Penzberg, Germany; for protocol details, see the manufacturer's instructions) gave the best reproducible amplification results concerning fragment size (1002000 bp) and DNA quantity for the ancient probes used in this study. Surprisingly, all DNA-free HIFI-DOP master mix controls showed positive amplification products. Using the modified primer set and the sensitive PCR system, the resulting DNA smear can be interpreted as contaminating bacterial DNA from polymerase preparation. To exclude contemporary human DNA contamination and to confirm the non-human contamination, we also labeled these products by nick translation and hybridized them in CGH experiments. CGH was performed as described previously with slight modifications (Tönnies et al. 2001
). Amplified test aDNAs and DNA-free HIFI-DOP master mix controls were labeled by nick translation using direct SpectrumGreen (test DNA)-conjugated deoxyuridine triphosphate (dUTP) (Vysis; Downers Grove, IL); contemporary male and female high-molecular reference DNAs were labeled by nick translation using SpectrumOrange-conjugated dUTP (Vysis). For each hybridization,
200 ng of labeled testaDNA, 200 ng reference DNA, and 12.5 µg Cot-1 DNA were mixed, ethanol precipitated, resuspended in hybridization mix containing 50% formamide, 2x SSC, and 10% dextran sulfate, denatured at 70C for 5 min, and applied to denatured male metaphase spreads at 37C for 3 days. After standard posthybridization washes, metaphases were analyzed using an epifluorescence microscope (Axiscope, Zeiss; Oberkochen, Germany) fitted with different single-band-pass filter sets for 4',6-diamidino-2-phenylindole [DAPI (blue)], SpectrumGreen (green), and SpectrumOrange (red) fluorescence (Figure 1). The microscope was equipped with an integrated high-sensitivity monochrome charge-coupled device camera (Hamamatsu; Shizuoka, Japan) for image acquisition. Image analysis and karyotyping were performed with the ISIS digital image analysis system (Metasystems; Altlussheim, Germany). Diagnostic thresholds of 0.80 and 1.25 were used for the identification of chromosomal underrepresentations (deletions) and overrepresentations (duplications) in the euchromatic chromosomal regions.
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Acknowledgments |
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Footnotes |
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Received for publication May 27, 2004; accepted September 2, 2004
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