Nucleic Acids Research, Vol. 19, No. 15 4315

PCR detection of five restriction site dimorphisms at the type I collagen loci COL1Al and COL1A2 R.Baker, J.Lynch, L.Ferguson, L.Priestley and B.Sykes* Collagen Genetics Group, University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK Source/Description: We have used PCR to amplify across five variable restriction sites at the loci COLlAl and COL1A2 which encode, respectively, the alI and a2 subunits of type I collagen. These sites are: Site A: An MspI site 26 kb upstream of the COLlAI gene (1). Site B: An RsaI site near the 5' end of the COLlAl gene (1). Site C: An EcoRI site within intron 12 of the COL1A2 gene (2). Site D: An MspI site in COL1A2 (3). Site E: An RsaI site towards the 3' end of COL1A2 (4). Primer sequences were derived as follows: Site A: By sequencing an 800 bp SailI-HindI fragment from cosmid CG102 which contains the variable MspI site. Site B: By sequencing in both directions from the BamHI site at the 5' end of subclone 2FC6. Site C: From the published sequence of the 5' end of intron 12 (5) and exon 13 (6). Site D: By subcloning and sequencing the 1.9 kb HindIII/XbaI fragment of p1A2E40. Site E: From the published sequences of the flanking exons 38 and 39 which flanked the variable site (6). Polymorphisms: Site A: Allele 1-0.1 + 0.1 kb, allele 2-0.2 kb Site B: Allele 1-0.8 + 0.2 kb, allele 2-1.0 kb Site C: Allele 1-0.8 + 0.7 kb, allele 2-1.5 kb Site D: Allele 1-0.85 + 0.45 kb, allele 2-1.3 kb. Constant band at 0.6 kb. Site E: Allele 1-0.8 + 0.1 kb, allele 2-0.9 kb Allele Frequencies: Site A: Al -0.76, A2-0.24. 182 UK Caucasian chromosomes. Site B: B1-0.88, B2-0.12. 164 UK Caucasian chromosomes. Site C: Ml -0.32, M2-0.68. 224 UK Caucasian chromosomes. Site D: NI -0.87, N2-0.13. 180 UK Caucasian chromosomes. Site E: 01-0.72, 02-0.28. 102 UK Caucasian chromosomes. Chromosomal Localisation: A, B: 17q21.31-22.15. C, D, E: 7q21.3 -22.1. Mendelian Inheritance: Co-dominant in more than 500 meioses from fifty families worldwide. PCR Primers: Site A: 5' CAGTTTATCAGATCCTAGTAGTTGAG 3' 5' CAATAGCCAGGCTGGTTTTGAACTCC 3' Site B: 5' ACAGGAAACTCTCACATA 3' 5' AGATCTCTGAGCATCTCT 3' Site C: 5' GGACTATGAGAGTCTGTTGA 3' 5' TGTTTGACCTGGAGTTCCAT 3' Site D: 5' TGTAAGTTTGTGAATACCAGT 3' 5' CATCAACTTCATAGTCCTTGG 3' Site E: 5' CTGCTGGAAGTCGTGGTGAT 3' 5' CACCAGGGAAACCAGTCATA 3' *

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Method: PCR conditions: 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.5 ltM each primer, 0.1 -1 tg genomic DNA, 1.5 units Ampli Taq (Cetus) in total volume 50 $1. 1 drop mineral oil (Sigma). Cycling conditions: Site A: 94°Cx3.0 min then 35 cycles of 55°Cx2.0 min, 72°Cx 1.0 min, 94°Cx 1.5 min. Site B: 93°Cx3.0 min then 30 cycles of 600C x 1.5 min, 700C x 1.0 min, 93°C x0.5 min. Site C: 940Cx3.0 min then 30 cycles of 60°Cx2.0 min, 720Cx 1.0 min, 930Cx0.5 min. Site D: 940Cx3.0 min then 30 cycles of 55°C x 1.5 min, 700C x 1.0 min, 93°C x0.5 min. Site E: 930Cx3.0 min then 30 cycles of 640Cx2.0 min, 93°C x0.5 min. All reactions were performed in an Autogene cycling water bath (Grant Instruments, Cambridge, UK). 8 11 aliquots of PCR product were digested with 10 units of the appropriate restriction enzyme and 1 Id of 10 xenzyme buffer for 2 hours at 37°C then run on gels containing either 2% Nusieve/1 % agarose (site A) or 1.5% agarose (other sites) and stained with ethidium bromide. Further Comments: These variable sites are used for prenatal diagnosis of dominantly-inherited osteogenesis imperfecta (7) which is consistently linked to one or other locus (8). PCR results show complete concordance with genotypes derived from Southern blotting. Primers and DNA from individuals of known genotype are available. Please address written enquiries to Dr. B.C.Sykes. Acknowledgements: We acknowledge the financial support of Action Research for the Crippled Child, Arthritis and Rheumatism Council and the Brittle Bone Society. Primer kits are sponsored by the EC Concerted Action on Heritable Connective Tissue Disorders. References: 1) Sykes et al. (1986) Lancet 69-72. 2) Tsipouras et al. (1983) J. Clin. Invest. 72, 1262-1267. 3) Grobler-Rabie et al. (1985) J. Med. Genet. 22, 182-186. 4) Grobler-Rabie et al. (1985) EMBO J. 4, 1745-1748. 5) Kuivaniemi et al. (1988) J. Biol. Chem. 263, 11407-11413. 6) de Wet et al. (1987) J. Biol. Chem. 262, 16032-16036. 7) Lynch et al. (1991) J. Med. Genet. 28, 145-150. 8) Sykes et al. (1991) Am. J. Hum. Genet. 46, 293-307. ii,

PCR detection of five restriction site dimorphisms at the type I collagen loci COL1A1 and COL1A2.

Nucleic Acids Research, Vol. 19, No. 15 4315 PCR detection of five restriction site dimorphisms at the type I collagen loci COL1Al and COL1A2 R.Baker...
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