CMSC423: Bioinformatic Algorithms, Databases and ToolsLecture 2Molecular biology primerAdmin...• Have you tried your glue accounts?• Issues/concerns/questions about class and policies?•Reading “assignment” - Chapter 1 in the book.The tree of lifehttp://www.fossilmuseum.net/Tree_of_Life/Domains_Archaea_Bacteria/DNA – the code of life• Purines A, G, caffeine•Pyrimidines C, T• Sugar backbone (ticker tape)• Double-stranded – allows replicationpictures from wikipediaDNA in the computer• FASTA/multi-FASTA file format>gi|110227054|gb|AE004091.2| Pseudomonas aeruginosa PAO1, complete genomeTTTAAAGAGACCGGCGATTCTAGTGAAATCGAACGGGCAGGTCAATTTCCAACCAGCGATGACGTAATAGATAGATACAAGGAAGTCATTTTTCTTTTAAAGGATAGAAACGGTTAATGCTCTTGGGACGGCGCTTTTCTGTGCATAACTCGATGAAGCCCAGCAATTGCGTGTTTCTCCGGCAGGCAAAAGGTTGTCGAGAACCGGTGTCGAGGCTGTTTCCTTCCTGAGCGAAGCCTGGGGATGAACGAGATGGTTATCCACAGCGGTTTTTTCCACACGGCTGTGCGCAGGGATGTACCCCCTTCAAAGCAAGGGTTATCCACAAAGTCCAGGACGACCGTCCGTCG•Parsers easy to write, also available in a variety of software librariesCentral dogmahttp://www.accessexcellence.org/RC/VL/GG/central.htmlAGGTACGCGTACCTGACAGGGenes, transcription, translation• DNA – RNA - Thymine replaced by Uracil (T-U)• The transcribed segments are called genes•AUG – start codon (also amino-acid Methionine)•UAA, UAG, UGA – stop codons • Genes are read in sets of 3 nucleotides during translation – 43 = 64 possible combinations•Each combination codes for one of 20 amino-acids – the building blocks for proteinsACCGUACCAUGUUA...AUAGGCUGAGCAAmino-acid translation tableGenes/proteins in the computer>gi|15596155|ref|NP_249649.1| basic amino acid, MKVMKWSAIALAVSAGSTQFAVADAFVSDQAEAKGFIEDSSLDLLLRNYYFNRDGKSGSGDRVDWTQGFLTTYESGFTQGTVGFGVDAFGYLGLKLDGTSDKTGTGNLPVMNDGKPRDDYSRAGGAVKVRISKTMLKWGEMQPTAPVFAAGGSRLFPQTATGFQLQSSEFEGLDLEAGHFTEGKEPTTVKSRGELYATYAGETAKSADFIGGRYAITDNLSASLYGAELEDIYRQYYLNSNYTIPLASDQSLGFDFNIYRTNDEGKAKAGDISNTTWSLAAAYTLDAHTFTLAYQKVHGDQPFDYIGFGRNGSGAGGDSIFLANSVQYSDFNGPGEKSWQARYDLNLASYGVPGLTFMVRYINGKDIDGTKMSDNNVGYKNYGYGEDGKHHETNLEAKYVVQSGPAKDLSFRIRQAWHRANADQGEGDQNEFRLIVDYPLSIL•Same FASTA/multi-FASTA but with bigger alphabetGenes/proteins in the computer gene complement(1043983..1045314) /gene="oprD" /locus_tag="PA0958" CDS complement(1043983..1045314) /gene="oprD" /locus_tag="PA0958" /note="Product name confidence: Class 1 (Function experimentally demonstrated in P. aeruginosa)" /codon_start=1 /transl_table=11 /product="Basic amino acid, basic peptide and imipenem outer membrane porin OprD precursor" /protein_id="AAG04347.1" /db_xref="GI:9946864"•GenBank file formatTranslation – complicationsAlternative splicing examplesProtein structure http://www.tulane.edu/~biochem/med/second.htmProtein structure• Primary structure – sequence• Secondary structure – structure motifs•Tertiary structure – 3D position of atoms•Quaternary structure – docking of proteinsProtein structure data (PDB format)ATOM 1 N MET A 1 20.020 28.662 42.801 1.00 51.80 N ATOM 2 CA MET A 1 20.598 29.950 42.438 1.00 52.13 C ATOM 3 C MET A 1 22.118 29.937 42.576 1.00 47.63 C ATOM 4 O MET A 1 22.660 29.623 43.636 1.00 49.97 O ATOM 5 CB MET A 1 20.009 31.073 43.293 1.00 51.36 C ATOM 6 CG MET A 1 20.331 32.468 42.765 1.00 51.13 C ATOM 7 SD MET A 1 21.406 33.373 43.921 1.00103.49 S ATOM 8 CE MET A 1 21.129 32.396 45.410 1.00 55.43 C ATOM 9 N LEU A 2 22.799 30.285 41.490 1.00 41.99 N ATOM 10 CA LEU A 2 24.249 30.178 41.424 1.00 37.25 CRECAP• DNA is a string formed with letters A, C, T, G (called nucleotides or bases)•DNA is double-stranded – allows replication: transfer of genetic “code” from parents to offspring•DNA is naturally oriented from 5’ to 3’ and the two strands are anti-parallel• If you know the sequence of one strand, you can obtain the sequence of the other by reverse-complementation5’ AGACCTAGTGCACGGCTACTACC 3’5’ CCATCATCGGCACGTGATCCAGA 3’Reverse5’ GGTAGTAGCCGTGCACTAGGTCT 3’ComplementRECAP• Central Dogma of molecular biology:– DNA – RNA (transcription)– RNA – Protein (translation)•The transcribed segments of DNA are called “genes”•Translation occurs in sets of 3 nucleotides – codons• Each codon encodes one of 20 amino-acids and 3 stop-codons•In eukaryotes the genes may be split into multiple exons, separated by introns: DNA segments that will not get translated• The protein is translated from an RNA representing the concatenation of the exons of the geneThe “new” biology• DNA is not the only heritable information– Epigenetic information: RNA molecules, DNA methylation patterns (affects coiling on DNA on histones)• Complex regulation patterns–Genes turn on other genes– Genes inhibit other genes–RNA interference – small RNA molecules can destroy specific transcripts (down-regulate production)Playing with DNABiologists can: • Cut the DNA – restriction enzymes (often palindromes) (Nobel prize – Arber, Nathans, Smith)• Attach “things” to DNA (either single or double-strand)•“Amplify” DNA – Polymerase Chain Reaction(Nobel prize – Mullis)5'GAATTC 5'---G AATTC---3' 3’CTTAAG 3'---CTTAA G---5' TAGGCACGTTGCAACTACGGC TGCAACGTPolymerase chain reaction (PCR)1. Denature2. Anneal (attachprimer)3. Extend4. RepeatHow does PCR work?• 1. Start: 1 double-stranded molecule• 1. Denature: 2 single-stranded molecules• 1. Anneal: 2 single-stranded molecules with primers attached•1. Extend: 2 double-stranded molecules – one “long” (L) strand and one “short” (S) (terminated at a primer)• 2. Start: 2 double-stranded molecules: L+S, L+S• 2. Denature: 2 x L strands, 2 x S strands • 2. Anneal: all strands with primers attached• 2. Extend: 2 double-stranded molecules: L+S, L+S, 2 double-stranded molecules: S+SS, S+SSSS – strand terminated at both ends with a primerPCR Recurrences•Ln, Sn, SSn - # of strands of each type at cycle n•Ln = Ln – 1 = 2•Sn = Sn – 1 + Ln – 1 = Sn – 1 + 2 = 2 * (n – 1) = O(n)•SSn = Sn-1 + 2 * SSn – 1 = O(2n)•The sequence between the primers (SS) is amplified exponentially – will quickly overtake the