CORE--MOLECULAR BIOLOGY DNA SEQUENCING CORE

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It is proposed that a DNA sequencing core facility be established fore the use of investigators associated with the Northeast Ohio Multipurpose Arthritis Center. Although the scope of molecular biology procedures used by laboratories associated with the Arthritis Center is quite broad and are project- specific, most laboratories would benefit from a core facility dedicated to DNA sequence determination. The rationale for this is that 1) DNA sequencing is required at some point in any molecular biology oriented experimental approach, and 2) it is not always cost effective for each laboratory to set up individual protocols, purchase equipment and train the necessary personnel. 3) DNA sequence analysis is labor and time intensive, but does not require specific knowledge of the larger project. 4) An on site sequencing facility could greatly enhance the overall productivity of Arthritis Center research laboratories by performing this task more efficiently and with greater precision than would be possible on an individual basis. 5) The core facility would free time for investigators to engage in more creative pursuits. The primary objective of the core would be to assist those investigators already engaged in molecular biology protocols. In order to be practical, it would not serve to perform and molecular biology techniques other than DNA sequence analysis. Investigators would be responsible for preparation of samples for sequencing in advance of submission. Protocols for sample preparation will be provided by the sequencing core in order to assure that the core facility will be efficiently utilized and to assure success. In general, investigators will be responsible for 1) ligation of DNA fragments into the polylinker (cloning site) of sequencing vectors commonly in use (i.e. M13 or Bluescript) for DNA sequence analysis by the Sanger dideoxynucleotide method. 2) In the case of M13, investigators will isolate M13 clones containing insert DNA and prepare single stranded template DNA. 3) In the case of plasmid vectors the investigator may either prepare single stranded template using a helper phage, or prepare double stranded plasmid suitable for sequencing. The core facility will then be responsible for performing the sequencing reactions, running sequencing gels, generating autoradiograms, reading DNA sequence and recording the sequence as a file that can be used for further analysis by the investigator. The second objective of the core would be to serve as a repository for genomic DNA from members of families afflicted with OA showing familial inheritance, and from patients with OA disease subsets. DNA from these individuals would be available to other investigators on a collaborative basis. Additionally, the core will function to screen this DNA bank for mutations in the type II collagen gene.