DNA sequencing methods文档.pptVIP

* * * * * * Massively parallel Ch-IP PCR, label with fluorescent dyes * Protein arrays for function Proteins immobilized, usually by virtue of a tag sequence (6 x his tag, biotin, etc.) Probe all proteins at once for a specific activity * Example of a protein microarray Proteins fused to GST with 6 x histidine tags, immobilized on Ni++ matrix Anti-GST tells how much protein is immobilized on surface Specific assays identify proteins with specific activities--calmodulin binding, phosphoinositide binding * DNA RNA protein genome “transcriptome” “proteome” (we have this) (we want these) * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Relationship between E-value and function Single domain proteins Multi-domain proteins E value greater than 10-10, similar structure but possibly different functions * Computer calls GNNTNNTGTGNCGGATACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCACCAC CACCACCACCCCATGGGTATGAATAAGCAAAAGGTTTGTCCTGCTTGTGAATCTGCGGAACTTATTTATGATCCAGAAAG GGGGGAAATAGTCTGTGCCAAGTGCGGTTATGTAATAGAAGAGAACATAATTGATATGGGTCCTAAGTGGCGTGCTTTTG ATGCTTCTCAAAGGGAACGCAGGTCTAGAACTGGTGCACCAGAAAGTATTCTTCTTCATGACAAGGGGCTTTCAACTGCA ATTGGAATTGACAGATCGCTTTCCGGATTAATGAGAGAGAAGATGTACCGTTTGAGGAAGTGGCANTCCANATTANGAGT TAGTGATGCAGCANANAGGAACCTAGCTTTTGCCCTAAGTGAGTTGGATAGAATTNCTGCTCAGTTAAAACTTCCNNGAC ATGTAGAGGAAGAAGCTGCAANGCTGNACANAGANGCAGNGNGANAGGGACTTATTNGANGCAGATCTATTGAGAGCGTT ATGGCGGCANGTGTTTACCCTGCTTGTAGGTTATTAAAAGNTCCCGGGACTCTGGATGAGATTGCTGATATTGCTAGAGC Raw data What does this sequence do? Cue up BLAST….. * MKCPYCKSRDLVYDRQHGEVFCKKCGSILATNLVDSELSRKTKTNDIPRYTKRIGEFTREKIYRLRKWQKKISSERNLVLAMSELRRLSGMLKLPKYVEEEAAYLYREAAKRGLTRRIPIETTVAACIYATCRLFKVPRTLNEIASYSKTEKKEIMKAFRVIVRNLNLTPKMLLARPTDYVDKFADELELSERVRRRTVDILRRANEEGITSGKNPLSLVAAALYIASLLEGERRSQKEIARVTGVSEMTVRNRYKELA Find the open reading frame(s) Translate it: * BLAST against (go to genomes page): -- Microbial genomes -- environmental sequences (genomes) Results: Distribution of hits: query sequence and po