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- 1. 2010/05/13 kaneko.satoko(at)ocha.ac.jp
- 2. Ensembl
- 3. Ensembl Ensembl (rat Olr1082 primer ) PCR primer/PCR Ensembl /Coding sequence (BLAT) / (9) BLAST (10) E‐value Primer‐BLAST (mouse Makorin1 ) Ensembl annotaNon (11) processed (pseudo)gene, unprocessed (pseudo)gene
- 4. Ensembl (olfactory receptor gene) 1 Olr1082 CDS query Olfactory receptor gene Olr1082 1 exon CDS 954bp (317a.a.) Reverse strand 954bp 1,500 Olr1082 primer
- 5. PCR primer PCR Polymerase Chain ReacNon DNA polymerase (DNA ) (genome DNA, cDNA) primer (dNTPs) 2 PCR primer A T C A G T C 20mer G ‐ C T ‐ A GC% 50% A ‐ T C ‐ G Tm 55‐65 C ‐ G primer 3' T ‐ A primer palindrome (palindrome TCCATGTATACGCCATGGA)
- 6. PCR PCR Denature: 95 (template 2 ) 30sec Annealing: 55‐65 (1 DNA primer annealing) 30sec Extension: 72 ( ) 1min/kb 30 Figure 8‐45b Molecular Biology of the Cell (© Garland Science 2008)
- 7. Ensembl h^p://www.ensembl.org/index.html Ensembl release 57 – March 2010 Search for rat Olr1082
- 8. Export data CDS
- 9. Coding sequence 1 Export data CDS [Output] [FASTA sequence] OpNons for FASTA sequence [Coding sequence] [Next >]
- 10. Coding sequence 2 [Text] FASTA format [HTML] [Compressed text (.gz)]
- 11. Coding sequence 3 [Text] FASTA format cds Olr1082 exon cds >ENSRNOG00000031936:ENSRNOT00000048054 cds:KNOWN_protein_coding CotEditor Macintosh HD/ /tg03/bin Olr1082.fasta +C ( ) +V
- 12. (BLAT) 1 BLAST/BLAT BLAT (Blast Like Alignment Tool) query/database 4,11‐mer (amino acid, DNA) EST
- 13. (9) BLAST BLAST (Basic Local Alignment Search Tool) alignment BLAST query database memo Arg Ser Val blastn DNA DNA Thr Ser Gly Tyr Val Arg blastp 5'-tacgtcaggta-3' blastx DNA 3'-atgcagtccat-5' Met Gln Ser tblastn DNA Cys Ser Pro Ala Val His tblastx DNA DNA PSI‐BLAST (PosiNon‐Specific Iterated, ) Primer‐BLAST (Primer3 and BLAST)
- 14. (BLAT) 2 Ra^us norvegicus (Select species ) dna database Search Tool BLAT RUN
- 15. (BLAT) view [Alignment LocaNons vs. Karyotype] [Alignment LocaNons vs. Query] query [Alignment Summary] query
- 16. (BLAT) Alignment LocaNons vs. Karyotype sort query
- 17. (BLAT) Alignment LocaNons vs. Query query
- 18. (BLAT) Alignment Summary query alignment (Stats Score, E‐value) query [S] [A] query HSP( ) Alignment alignment query alignment query [G] exon [S]+
- 19. (10):E‐value query query (Expect value) (ex. 0.0, e‐100 ) query E‐value query ‐ *E‐value *
- 20. (BLAT) Alignment Summary [C] ConNg view
- 21. (BLAT) primer 1 6 query hit query Olr1082 [A] Olr1082 (20bp ) (primer )
- 22. (BLAT) primer 2 Olr1082 8 Ensembl gene Olr : cds Olr1082 primer
- 23. Primer‐BLAST h^p://www.ncbi.nlm.nih.gov/tools/primer‐blast/ primer Primer3 BLAST (accession, gi "GenInfo IdenNfier", FASTA ) primer primer PCR primer primer Tm ( Forward Reverse )
- 24. Primer‐BLAST –exon/intron‐ Exon junc7on span: primer exon exon (exon exon primer mRNA ) Exon junc7on match: exon bp Intron inclusion: PCR 1 intron Intron length range: PCR intron
- 25. Primer‐BLAST –parameter‐ Specificity check: primer Organism: template ( ) Database: (template DNA Genome Refseq RNA ) non‐redundant Primer specificity stringency: mismatch Misprimed product size deviaNon: Splice vairant handling: template splice variant
- 26. Primer‐BLAST –Advanced parameters‐ BLAST hit BLAST E‐value primer pair PCR Tm primer ( ) primer GC primer 3' 5bp G C palindrome SNP Repeat primer PCR
- 27. Primer‐BLAST query primer primer pair primer pair BLAST query
- 28. Makorin1’ Makorin RING zinc finger Makorin Makor + in Makor in Zinc Finger RING The source of RING (Gray et al. 2000 Genomics. 66:76‐86.) 6 22.56kb 8 exon 22.56kb Reverse strand 8 exons, CDS 1446bp (481a.a.) Makorin1 CDS
- 29. Makorin1 8 exons, CDS 1446bp (481a.a.) CDS ID: ENSMUS BLAST Makorin1 CDS Chromosome Gene locaNon Gene structure, feature Makorin1 6 B1 exon‐intron
- 30. 1 1) Search Mouse for Makorin 2) Makorin1 CDS makorin.fasta [Macintosh HD/ /tg03/bin] Makorin1 4 transcript Makorin1 CDS
- 31. Ensembl annotaNon UniProtKB (SwissProt and TrEMBL) Havana (Human And Vertebrate Analysis aNd AnnotaNon) annotaNon, splice variant pseudogene annotaNon Ensembl annotaNon Vega ( E ) annotaNon manual annotaNon
- 32. 3) Makorin1 CDS query BLAT (Selected species Mus musculus ) 4) Makorin1 5) exon‐intron
- 33. (11) processed (pseudo)gene, unprocessed (pseudo)gene a a' unprocessed gene duplicated gene a mRNA splicing a'' processed gene pseudogene premature stop codon (inserNon) (deleNon)
- 34. 3 Makorin 4 Chromosome Gene locaNon Gene structure Makorin1 6 B1 exon‐intron Makorin2 6 E3 exon‐intron Makorin3 7 C processed, intact Makorin1‐p1 5 E2 processed, premature stop Makorin1‐p2 3 G3 processed, fragmented Makorin1‐p3 5 F processed, fragmented Makorin1‐p4 13 C1 processed, fragmented Makorin1 Makorin2 ( Makorin2 ) 20% Makorin2 hit
- 35. Ensembl Dotplot