Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis.
BACKGROUND
In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature.


METHODS
Synchronized cultured cells were fixed with methanol/acetic acid to prepare chromosome spreads in teflon-coated diagnostic well-slides. Apart from the slide format and the chromosome spreading everything was done essentially according to standard protocols. Hybridization targets were detected in situ with padlock probes, which were ligated and amplified using target primed rolling circle DNA synthesis, and detected by fluorescence labeling.


RESULTS
An optimized protocol for the spreading of condensed metaphase chromosomes in teflon-coated diagnostic well-slides was developed. Applying this protocol we generated specimens for target primed rolling circle DNA synthesis of padlock probes recognizing a 40 nucleotide sequence in the male specific repetitive satellite I sequence (DYZ1) on the Y-chromosome and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a) gene positioned on the long arm of chromosome 6. These targets were detected with good efficiency, but the efficiency on other target sites was unsatisfactory.


CONCLUSIONS
Our aim was to test the applicability of the method used on mitochondrial DNA to the analysis of nuclear genomes, in particular as represented by metaphase spreads. An optimized protocol for chromosome spreading in diagnostic well-slides was used for the detection of circularized padlock probes amplified by target primed rolling circle DNA synthesis from condensed metaphase chromosomes. We were able to detect a 40 nucleotide sequence in the male specific repetitive satellite I sequence and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a) gene. Our overall conclusion is that whilst this type of reaction indeed can be brought to work on nuclear genomes, including metaphase chromosomes, the total efficiency of this multistep reaction is at present relatively low (1-10% of target sites picked up), meaning that it is best suited for the detection of targets that exist in multiple copies per cell. Changing this will require substantial efforts to systematically increase the efficiency in each step.
Lohmann
Jakob S
JS
Institute of Pathology, Aarhus University, 8000 Aarhus C, Denmark. jlohm@as.aaa.dk
Stougaard
Magnus
M
Koch
Jørn
J
D002467:Cell Nucleus / Q000378:metabolism; D002875:Chromosomes / Q000737:chemistry / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics*; D004270:DNA, Circular / Q000032:analysis / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008214:Lymphocytes / Q000378:metabolism; D008297:Male; D008677:Metaphase / Q000235:genetics*; D015336:Molecular Probe Techniques; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020401:Primed In Situ Labeling / Q000379:methods; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA / Q000379:methods*
D015342:DNA Probes; D004270:DNA, Circular