Single base discrimination of CENP-B repeats on mouse and human Chromosomes with PNA-FISH.
The satellite repeat structure of the mammalian centromere contains the CENP-B protein binding site. Using the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH), we show by direct PNA-DNA binding that all detectable CENP-B sites in a mammalian genome might have the same sequence. Two species-specific PNA 17-mers, pMm and pMc, were identified from CENP-B binding sites of Mus musculus and M. caroli, respectively. Fluorescence in situ hybridization confirmed that pMc hybridized to M. caroli centromeres only; however, pMm cross-hybridized to M. musculus and human centromeres. By using a series of CENP-B PNA 17-mers containing 1, 2, 3, 5, and 7 base-pair mismatches to their DNA counterparts, we further demonstrate that PNA-FISH can discriminate between two CENP-B DNA sequences that differ by a single base-pair in mouse and human centromeres, suggesting the degree of conservation of CENP-B sequences throughout the genome. In comparison with DNA oligonucleotides, PNA oligomers demonstrate the higher sequence specificity, improved stability, reproducibility, and lower background. Therefore, PNA oligomers have significant advantages over DNA oligonucleotide probes in analyzing microsatellites in a genome.
Chen
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C
Harvard Institutes of Human Genetics, Harvard Medical School, Beth Israel Deaconess Medical Center, 4 Blackfan Circle, Boston, Massachusetts 02115, USA.
Hong
Y K
YK
Ontiveros
S D
SD
Egholm
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M
Strauss
W M
WM
D000818:Animals; D001324:Autoantigens; D020029:Base Pairing; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics*; D017931:DNA Primers; D004268:DNA-Binding Proteins; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D020135:Peptide Nucleic Acids; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity; D013045:Species Specificity
D001324:Autoantigens; C497926:CENPB protein, human; C497927:Cenpb protein, mouse; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D017931:DNA Primers; D004268:DNA-Binding Proteins; D020135:Peptide Nucleic Acids