基因,蛋白,免疫 课程2.ppt

DNA sequencing Principles and Practice and the Human Genome Project. The sequencing reaction All current sequencing methods are based on that developed by Fred Sanger (MRC Lab Cambridge) Synthesis of DNA by a DNA polymerase from a single stranded template. Synthesis primed by a specific oligonucleotide. The reactions are carried out in a mixture of normal deoxynucleotides and one chain terminating di-deoxynucleotide giving rise to a population of molecules of different sizes. The synthesised strand is labelled. The population of synthesised molecules is then separated on the basis of length. Sequencing using capillary electrophoresis Introduction of capillary electrophoresis. The products of the sequencing reaction pass down a fine capillary (75 μ I.d. 40 cm long) filled with a linear polyacrylamide polymer. The capillaries are scanned using confocal laser optics. Each machine can contain upto 384 capillaries. Advantages are high capacity, high sensitivity (requires small amount of sequencing product), long reads (1000 basas), speed (2 hour turn around time). Use fluorescent dye terminators. Human Genome Project Two competing groups International human genome consortium Celera Genomics (Craig Venter) Draft human genome completed October 2000. Based on sequencing from a limited number of individuals. Blood, sperm and immortalised cell lines One article published by each group February 2001 Venter Science 291 1305-1351 IHGSC Nature 409 860 920 Technical Innovation Key to Sequencing the Human genome Summary Four- colour fluorescence-based sequence detection. Improved fluorescent dyes. Dye labelled terminators. Polymerases specifically designed for sequencing. Cycle sequencing Capillary gel electrophoresis Development of powerful software for recognising and merging overlapping sequences. Genetic maps All the sequence data was hung onto preexisting maps of the human genome. Cytogenetic map (map spacing 0.5 Mb or more) Restriction map (map spacing 100 kb) Clone conti