Abstract
The solid-phase minisequencing method (Syvänen et al. 1990) allows accurate quantative determination of the ratio between two DNA or RNA sequences that are present as a mixture in a sample and differ from each other only by a single nucleotide. Here, we present another application of the minisequening method, the determination of the gene copy number in a genome. The copy number of a marker gene aspartyl glucosaminidase (AGA) located at 4qter, was determined in three patients with a chromosomal alteration involving the distal region of 4q. For the minisequencing assay an equal amount of DNA from a patient homozygous for a mutation in the AGA gene was added to the DNA samples concerned. The relative amount of the normal sequence determined in each combined sample gives the copy number of the AGA gene. Fluorescence in situ hybridization (FISH), applied in parallel as a control, produced concordant results with solid-phase minisequencing in each case. As the potential of the minisequencing lies in automation, it could be a useful tool in the screening of monosomies, trisomies or loss of heterozygosity in diagnostics.
Similar content being viewed by others
References
Aula P, Astrin KH, Francke U, Desnick RJ (1984) Assignment of the structural gene encoding human aspartylglucosaminidase to the long arm of chromosome 4 (4q21→ter). Am J Hum Genet 36:1215–1224
Bell GI, Karam JH, Rutter WJ (1981) Polymorphic DNA region adjacent to the 5′ end of the human insulin gene. Proc Natl Acad Sci USA 78:5759–5763
Engelen J, Hamers A, Schrander-Stumpel C, Mulder H, Poorthuis B (1992) Assignment of the aspartylglucosaminidase gene (AGA) to 4q33→q35 based on decreased activity in a girl with a 46,XX,del(4)(q33) karyotype. Cytogenet Cell Genet 60:208–209
Grön K, Aula P, Peltonen L (1990) Linkage of aspartylglucosaminuria (AGU) to marker loci on the long arm of chromosome 4. Hum Genet 85:233–236
Hietala M, Grön K, Syvänen A-C, Peltonen L, Aula P (1993) Prospects of carrier screening of aspartylglucosaminuria in Finland. Eur J Hum Genet 1:296–300
Ikonen E, Baumann M, Grön K, Syvänen E-C, Enomaa N, Halila R, Aula P, Peltonen L (1991) Aspartylglucosaminuria cDNA encoding human aspartylglucosaminidase and the missense mutation causing the disease. EMBO J 10:51–58
Ikonen E, Manninen T, Peltonen L, Syvänen A-C (1992) Quantitative determination of rare mRNA species by PCR and solidphase minisequencing. PCR Methods Applications 1:234–240
Jalanko A, Kere J, Savilahti E, Schwartz M, Syvänen AC, Ranki M, Söderlund H (1992) Screening for defined cystic fibrosis mutations by solid-phase minisequencing. Clin Chem 38:39–43
Kallioniemi A, Kallioniemi OP, Waldman FM, Chen LC, Yu LC, Fung YKT, Smith HS, Pinkel D, Gray JW (1992) Detection of retinoblastoma gene copy number in metaphase chromosomes and interphase nuclei by fluorescence in situ hybridization. Cytogenet Cell Genet 60:190–193
Lawrence JB, Villnave CA, Singer RH (1988) Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell 52:51–61
Lichter P, Tang CJ, Call K, Hermanson G, Evans GA, Housman D, Ward DC (1990) High resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247:64–69
Lubin MB, Elashoff JD, Wang S-J, Rotter J, Toyoda H (1991) Precise gene dosage determination by polymerase chain reaction: theory, methodology and statistical approach. Mol Cell Probes 5:307–317
Pinkel D, Straume T, Gray JW (1986) Cytogenetic analysis using quantification, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83:2934–2938
Seabright M (1971) A rapid banding technique for human chromosomes. Lancet 2:971–972
Suomalainen A, Kollmann, Octave JN, Söderlund H, Syvänen A-C (1993a) Quantification of mitochondrial DNA carrying the tRNA Lys8344 point mutation in myoclonus epilepsy and raggedfiber disease. Eur J Hum Genet 1:88–95
Suomalainen A, Majander A, Pihko H, Peltonen L, Syvänen A-C (1993b) Quantification of the tRNA Leu3243 point mutation of the mitochondrial DNA in MELAS patients and its effects on mitochondrial transcription. Hum Mol Genet 2:525–534
Syvänen A-C, Aalto-Setälä K, Harju L, Kontula K, Söderlund H (1990) A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 8:684:692
Syvänen A-C, Ikonen E, Manninen T, Bengström M, Söderlund H, Aula P, Peltonen L (1992) Convenient and quantitative deterination of the frequency of a mutant allele using solid-phase minisequencing: application to aspartylglucosaminuria in Finland. Genomics 12:590–595
Xeros N (1962) Deoxyriboside control and synchronization of mitosis. Nature 194:682–683
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Laan, M., Grön-Virta, K., Salo, A. et al. Solid-phase minisequencing confirmed by FISH analysis in determination of gene copy number. Hum Genet 96, 275–280 (1995). https://doi.org/10.1007/BF00210406
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00210406