Williams C J, Harrison D A, Hopkinson I, Baldwin C T, Ahmad N N, Ala-Kokko L, Korn R M, Buxton P G, Dimascio J, Considine E L
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Jefferson Medical College, Philadelphia, Pennsylvania 19107.
Hum Mutat. 1992;1(5):403-16. doi: 10.1002/humu.1380010510.
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.
本文描述了从聚合酶链反应(PCR)扩增的基因组DNA中对人II型前胶原(COL2A1)基因进行直接测序的方法。使用内含子引物对COL2A1基因的32个区域进行不对称扩增,这些引物经过特别选择,用于扩增跨越500至800 bp的序列区域,该区域编码一个或多个外显子及其伴随的间隔序列。然后设计用于PCR产物双脱氧核苷酸测序的引物,以提供完整的外显子序列信息,并确保获得内含子-外显子剪接连接序列数据。在自动工作站上进行扩增和测序反应,以方便处理多个DNA模板。该方法允许对每个二倍体基因组中COL2A1基因的每个等位基因超过25,000 bp进行高效测序。我们使用这种方法对从42名无关个体的血液中分离的DNA中的COL2A1序列进行了比较分析,并在该基因中鉴定出21个中性序列变体。通过包括限制性酶切消化在内的独立检测方法确认了序列变异。本文所述的序列变异对于鉴定II型前胶原基因的单倍型非常重要,这将有助于确定软骨组织疾病的遗传病因。