title: Detection of sequence variants in the gene for human type II procollagen (COL2A1) by direct sequencing of polymerase chain reaction-amplified genomic DNA.
abstract: The direct sequencing of the human type II procollagen (COL2A1) gene from polymerase chain reaction (PCR)-amplified genomic DNA is described. Thirty-two regions of the COL2A1 gene were asymmetrically amplified with intron primers which were specifically chosen to amplify a region spanning 500 to 800 bp of sequence encoding one or more exons and their accompanying intervening sequences. Primers for dideoxynucleotide sequencing of the PCR products were then designed to provide complete exon sequence information and to insure that intron:exon splice junction sequence data would be obtained. Amplification and sequencing reactions were performed on an automated workstation to facilitate the handling of multiple DNA templates. The procedure allowed efficient sequencing of over 25,000 bp of each allele of the COL2A1 gene per diploid genome. We used this method for the comparative analyses of COL2A1 sequences in DNA isolated from the blood of 42 unrelated individuals and we identified 21 neutral sequence variants in the gene. The sequence variations were confirmed by independent assays, including restriction enzyme digestion. The sequence variants described here will be important for identifying haplotypes of the type II procollagen gene that will be useful in defining a genetic etiology for diseases of cartilaginous tissues.
authors.affiliation: Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Philadelphia, Pennsylvania 19107.
authors.forename: C J
authors.initials: CJ
authors.lastname: Williams
authors.forename: D A
authors.initials: DA
authors.lastname: Harrison
authors.forename: I
authors.initials: I
authors.lastname: Hopkinson
authors.forename: C T
authors.initials: CT
authors.lastname: Baldwin
authors.forename: N N
authors.initials: NN
authors.lastname: Ahmad
authors.forename: L
authors.initials: L
authors.lastname: Ala-Kokko
authors.forename: R M
authors.initials: RM
authors.lastname: Korn
authors.forename: P G
authors.initials: PG
authors.lastname: Buxton
authors.forename: J
authors.initials: J
authors.lastname: Dimascio
authors.forename: E L
authors.initials: EL
authors.lastname: Considine
mesh_terms: D000483:Alleles; D001483:Base Sequence; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics; D015342:DNA Probes; D005787:Gene Frequency; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011347:Procollagen / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D015342:DNA Probes; D011347:Procollagen; D004247:DNA