A strategy for the amplification, purification, and selection of M13 templates for large-scale DNA sequencing.
A strategy addressing various aspects of generating and processing DNA sequencing templates is described. The strategy is designed for large-scale DNA sequencing projects and involves procedures for the amplification, purification, and selection of M13 DNA templates. The amplification is based on growth in liquid culture, the purification is based on a novel thermoextraction method which replaces phenol extraction and ethanol precipitation, and the template selection is based on hybridization of gridded template arrays on membranes with chemiluminescent probes. All steps are currently carried out manually but the use of standardized formats (96-well format) throughout the entire strategy provides for possible transition to partial or full automation in the future. The strategy has been developed and tested within the ongoing project of sequencing the class II region of the human major histocompatibility complex but it should be equally applicable to any large-scale DNA sequencing project.
Beck
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S
Imperial Cancer Research Fund, London, England.
Alderton
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RP
D002498:Centrifugation; D002623:Chemistry Techniques, Analytical; D004247:DNA / Q000032:analysis* / Q000235:genetics* / Q000302:isolation & purification; D006358:Hot Temperature; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic
D004247:DNA