Abstract
It is demonstrated that either general staining of the centromeric regions of all primate chromosomes, or selective staining of the centromeric region of specific chromosomes, may be obtained in preparations of metaphase chromosomes by probing specifically for different regions within the alpha satellite DNA monomer. In order to exploit observed patterns of sequence variation within the monomer for this purpose, we have developed two new DNA analysis methods. In PRimed IN Situ labelling (PRINS), synthetic oligonucleotides derived from subsections of the monomer are hybridized to the chromosomes. The oligonucleotides then serve as primers for the in situ incorporation of biotin-labelled nucleotides catalysed by Klenow polymerase. Incorporated biotin is visualized with fluorescein isothiocyanate-labelled avidin (FITC-avidin). In Primed Amplification Labelling (PAL), biotin-labelled hybridization probes are produced in a polymerase chain reaction (PCR, Saiki et al. 1985), in which two synthetic oligonucleotide primers anneal within the same monomer. With the right choice of primers libraries of labelled probes derived from most monomers present as templates are produced. If DNA from a specific chromosome is used as template, then the resulting probe mixture gives stronger and more chromosome-specific signals in in situ hybridization experiments than does a cloned alpha satellite DNA probe derived from the same chromosome. The results obtained indicate that the alpha-repeat monomer is composed of regions with different degrees of chromosome specificity.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bachvarov DR, Markov GG, Ivanov IG (1987) Sequence heterogeneity of the human alphoid satellite DNA and thermal stability of mismatched alphoid DNA duplexes. Int J Biochem 19:963–971
Cremer T, Landegent J, Brückner A, Scholl HP, Schardin M, Hager HD, Devilee P, Pearson P, van der Ploeg M (1986) Detection of chromosome aberrations in the human nucleus by visualization of specific target DNAs with radioactive and nonradioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L 1.84. Hum Genet 74:346–352
Crolla JA, Llerena Jr JC (1988) A mosaic 45,X/46,X, r(?) karyotype investigated with X and Y centromere-specific probes using a non-autoradiographic in situ hybridization technique. Am J Med Genet 81:81–84
Devilee P, Slagboom P, Cornelisse CJ, Pearson PL (1986) Sequence heterogeneity within the human alphoid repetitive DNA family. Nucleic Acids Res 14:2059–2073
Donehower L, Furlong C, Gillespie D, Kurnit D (1980) DNA sequence of baboon highly repeated DNA: Evidence for evolution by unequal crossovers. Proc Natl Acad Sci USA 77:2129–2133
Fantes J, Gosden J, Piper J (1988) Use of an alphoid satellite sequence to locate the X-chromosome automatically, with particular reference to identification of the fragile X. Cytogenet Cell Genet 48:142–147
Goss SJ, Harris H (1977) Gene transfer by cell fusion. Statistical mapping of human X-chromosome by analysis of radiation induced gene segregation. J Cell Sci 25:17
Gregersen N, Koch J, Kølvraa S, Petersen KB, Bolund L (1987) Improved methods for the detection of unique sequences in Southern blots of mammalian DNA by non-radioactive biotinylated DNA hybridization probes. Clin Chim Acta 169:267–280
Koch J, Kølvraa S, Bolund L (1986) An improved method for labelling of DNA probes by nicktranslation. Nucleic Acids Res 14:7132
Koch J, Kølvraa S, Gregersen N, Petersen KB, Bolund L (1988) PRINS and PAL — two new techniques for the visualization of specific DNA sequences. Genome 30, suppl 1:446
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory Press, NY
Pike LM, Carlisle A, Newell C, Hong S, Musich R (1986) Sequence and evolution of Rhesus Monkey alphoid DNA. J Mol Evol 23:127–137
Pinkel D, Straume T, Gray JW (1986) Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83:2934–2938
Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354
Singer MF (1982) Highly repeated sequences in mammalian genomes. Int Rev Cytol 76:67–112
Strauss F, Varshavsky A (1984) A protein binds to a satellite DNA repeat at three specific sites that would be brought into mutual proximity by DNA folding in the nucleosome. Cell 37:889–901
Waldren C, Martin J, Sutherland J, Cram S (1984) Use of somatic cell hybrids for quantitation of mutagenesis: reduction in background mutants by fluorescence-activated cell sorting (FACS). Cytometry 5:584–588
Waye JS, Willard HF (1985) Chromosome specific alpha satellite DNA: Nucleotide sequence analysis of the 2.0 kilobase pair repeat from the human X-chromosome. Nucleic Acids Res 13:2731–2743
Waye JS, Willard HF (1987) Nucleotide sequence heterogeneity of alpha satellite repetitive DNA: a survey of alphoid sequences from different human chromosomes. Nucleic Acids Res 15:7549–7567
Willard HF, Waye JS (1987) Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genet 3:192–198
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Koch, J.E., Kølvraa, S., Petersen, K.B. et al. Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ. Chromosoma 98, 259–265 (1989). https://doi.org/10.1007/BF00327311
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00327311