Introduktion til Bioinformatik Hold 01 Oktober 2010 Introduktion
Introduktion til Bioinformatik Hold 01 Oktober 2010
Introduktion Rasmus Wernersson, Lektor Anders Gorm Pedersen, Docent Center for Biologisk Sekvensanalyse, DTU
Oversigt • Taksonomi • DNA Data & Databaser • Protein struktur • Alignment Metoder • Pairwise + Multiple • BLAST (søgning) • Fylogenetiske træer • Py. MOL (3 D visualisering) Opsamlende øvelse Malaria vaccine
Øvelserne er det primære
Kursusplan på vores wiki
Background information On evolution and sequences
Classification: Linnaeus Carl Linnaeus 1707 -1778
Classification: Linnaeus • Hierarchical system – – – – Kingdom Phylum Class Order Family Genus Species
Classification depicted as a tree
No “mixed” animals Source: www. dr. dk/oline
Classification depicted as a tree Species Genus Family Order Class
Comparison of limbs Image source: http: //evolution. berkeley. edu
Theory of evolution Charles Darwin 1809 -1882
Phylogenetic basis of systematics • Linnaeus: Ordering principle is God. • Darwin: Ordering principle is shared descent from common ancestors. • Today, systematics is explicitly based on phylogeny.
Natural Selection: Darwin’s four postulates • More young are produced each generation than can survive to reproduce. • Individuals in a population vary in their characteristics. • Some differences among individuals are based on genetic differences. • Individuals with favorable characteristics have higher rates of survival and reproduction. • • • Evolution by means of natural selection Presence of ”design-like” features in organisms: Quite often features are there “for a reason”
Evolution at the sequence level
About DNA • DNA contains the recipes of how to make protein / enzymes. • Every time a cells divides it’s DNA is duplicated, and each daughter cell gets a copy.
The DNA alphabet • The information in the DNA is written in a four letter code: A, T, G, C. • The DNA can be “sequenced” and the result stored in a computer file. • ATGGCCCTGTGGAT
DNA is always written 5’ 3’ Ribose 3’ 5 4 1 3 2 5’ Deoxyribose 5 4 1 3 2 5’ AGCC 3’ 3’ TCGG 5’ 5’ 5’ ATGGCCAGGTAA 3’ DNA backbone: http: //en. wikipedia. org/wiki/DNA (Deoxy)ribose: http: //en. wikipedia. org/ 3’
Can DNA be changed? • ATGGCCCTGTGGATGCG
Can DNA be changed? • ATGGCCCTGTGGATGCG • ATGGCCCTATGGATGCG
A history of mutations ATGGCAATGTGGATGCA ATGGCCCCGTGGAACCG ATGTCCCCGTGGATGCG ATGGCCCTGTGGATGCG Time ATGGCCCTGTGTATGCG
“DNA alignment” • Species 1: • Species 2: • Species 3: ATGGCAATGTGGATGCA ATGGCCCCGTGGAACCG ATGTCCCCGTGGATGCG 6 3 5
Real life example: Alignment • Insulin from 7 different species • • Homo: Pan: Sus: Ovis: Canis: Mus: Gallus: ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGACCCAGCCGCAGCCTTTGTGAA ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGTGCTGCTGGCCCTCTGGGGACCTGACCCAGCCTCGGCCTTTGTGAA ATGGCCCTGTGGACGCGCCTCCTGCCCCTGCTGGCCCTCTGGGCGCCCCGGCCCAGGCCTTCGTGAA ATGGCCCTGTGGACACGCCTGGTGCCCCTGCTGGCACTCTGGGCCCCCGGCCCACGCCTTCGTCAA ATGGCCCTCTGGATGCGCCTCCTGCCCCTGCTGGCCCTCTGGGCGCCCACCCGAGCCTTCGTTAA ATGGCCCTGTTGGTGCACTTCCTACCCCTGCTGGCCCTGCTTGCCCTCTGGGAGCCCAAACCCAGGCTTTTGTCAA ATGGCTCTCTGGATCCGATCACTGCCTCTTCTGGCTCTCCTTGTCTTTTCTGGCCCTGGAACCAGCTATGCAGCTGCCAA
Real life example: Tree
Interpretation of Multiple Alignments Conserved features assumed to be important for functionality For instance: conserved pairs of cysteines indicate possible disulphide bridge
Sequences are related • Darwin: all organisms are related through descent with modification • Prediction: similar molecules have similar functions in different organisms Protein synthesis carried out by very similar RNA-containing molecular complexes (ribosomes) that are present in all known organisms
Sequences are related, II Related oxygenbinding proteins in humans
DNA as Biological Information Rasmus Wenersson
Overview • Learning objectives – About Biological Information – A note about DNA sequencing techniques and DNA data – File formats used for biological data – Introduction to the Gen. Bank database
Information flow in biological systems
DNA sequences = summary of information Ribose 3’ 5 4 1 3 2 5’ Deoxyribose 5 4 1 3 2 5’ AGCC 3’ 3’ TCGG 5’ 5’ 5’ ATGGCCAGGTAA 3’ DNA backbone: http: //en. wikipedia. org/wiki/DNA (Deoxy)ribose: http: //en. wikipedia. org/ 3’
PCR Melting 96º , 30 sec 35 cycles Annealing ~55º, 30 sec Extension 72º , 30 sec Animation: http: //depts. washington. edu/~genetics/courses/genet 371 b-aut 99/PCR_contents. html
PCR Animation: http: //www. people. virginia. edu/~rjh 9 u/pcranim. html PCR graph: http: //pathmicro. med. sc. edu/pcr/realtime-home. htm
Gel electrophoresis • DNA fragments are seperated using gel electrophoresis – Typically 1% argarose – Colored with Et. Br or Zybr. Green (glows in UV light). – A DNA ”ladder” is used for identification of known DNA lengths. - + Gel picture: http: //www. pharmaceutical-technology. com/projects/roche/images/roche 3. jpg PCR setup: http: //arbl. cvmbs. colostate. edu/hbooks/genetics/biotech/gels/agardna. html
The Sanger method of DNA sequencing } OH Terminator X-ray sequenceing gel Images: http: //www. idtdna. com/support/technical/Technical. Bulletin. PDF/DNA_Sequencing. pdf
Automated sequencing • The major break-through of sequencing has happended through automation. • Fluorescent dyes. • Laser based scanning. • Capillary electrophoresis • Computer based basecalling and assembly. Images: http: //www. idtdna. com/support/technical/Technical. Bulletin. PDF/DNA_Sequencing. pdf
Handout exercise: ”base-calling” • Handout: Chromotogram • Groups of 2 -3. • Tasks: – Identify “difficult” regions – Identify “difficult” sequence stretches. – Try to estimate the best interval to use.
Biological data on computers • The Gen. Bank database • File formats – FASTA – Gen. Bank
NCBI Gen. Bank • Gen. Bank is one of the main internaltional DNA databases. • Gen. Bank is hosted by NCBI: National Center for Biotechnology Information. • Gen. Bank has exists since 1982. • The database is public - no restrictions on the use of the data within.
FASTA format >alpha-D ATGCTGACCGACTCTGACAAGAAGCTGGTCCTGCAGGTGTGGGAGAAGGTGATCCGCCAC CCAGACTGTGGAGCCGAGGCCCTGGAGAGGTGCGGGCTGAGCTTGGGGAAACCATGGGCA AGGGGGGCGACTGGGAGCCCTACAGGGCTGCTGGGGGTTGTTCGGCTGGGGGTCAG CACTGACCATCCCGCAGCTGTTCACCACCTACCCCCAGACCAAGACCTACTTCC CCCACTTCGACTTGCACCATGGCTCCGACCAGGTCCGCAACCACGGCAAGAAGGTGTTGG CCGCCTTGGGCAACGCTGTCAAGAGCCTGGGCAACCTCAGCCAAGCCCTGTCTGACCTCA GCGACCTGCATGCCTACAACCTGCGTGTCGACCCTGTCAACTTCAAGGCGGGGGAC GGGGGTCAGGGGCCGGGGAGTTGGGGGCCAGGGACCTGGTTGGGGATCCGGGGCCATGCC GGCGGTACTGAGCCCTGTTTTGCCTTGCAGCTGCTGGCGCAGTGCTTCCACGTGGTGCTG GCCACACACCTGGGCAACGACTACACCCCGGAGGCACATGCTGCCTTCGACAAGTTCCTG TCGGCTGTGTGCACCGTGCTGGCCGAGAAGTACAGATAA >alpha-A ATGGTGCTGTCTGCCAACGACAAGAGCAACGTGAAGGCCGTCTTCGGCAAAATCGGCGGC CAGGCCGGTGACTTGGGTGGTGAAGCCCTGGAGAGGTATGTGGTCATCCGTCATTACCCC ATCTCTTGTCTGTGACTCCATCTGCCCCCATACTCTCCCCATAACTG TCCCTGTTCTATGTGGCCCTGGCTCTGTCTCATCTGTCCCCAACTGTCCCTGATTGCCTC TGTCCCCCAGGTTGTTCATCACCTACCCCCAGACCAAGACCTACTTCCCCCACTTCGACC TGTCACATGGCTCCGCTCAGATCAAGGGGCACGGCAAGAAGGTGGCGGAGGCACTGGTTG AGGCTGCCAACCACATCGATGACATCGCTGGTGCCCTCTCCAAGCTGAGCGACCTCCACG CCCAAAAGCTCCGTGTGGACCCCGTCAACTTCAAAGTGAGCATCTGGGAAGGGGTGACCA GTCTGGCTCCCCTCCTGCACACACCTCTGGCTACCCCCTCACCCCCTTGCTCACC ATCTCCTTTTGCCTTTCAGCTGCTGGGTCACTGCTTCCTGGTGGTCGTGGCCGTCCACTT CCCCTCTCTCCTGACCCCGGAGGTCCATGCTTCCCTGGACAAGTTCGTGTGTGCCGTGGG CACCGTCCTTACTGCCAAGTACCGTTAA (Handout)
Gen. Bank format • Originates from the Gen. Bank database. • Contains both a DNA sequence and annotation of feature (e. g. Location of genes). (handout)
Gen. Bank format - HEADER LOCUS DEFINITION ACCESSION VERSION KEYWORDS SOURCE ORGANISM REFERENCE AUTHORS TITLE JOURNAL PUBMED COMMENT CMGLOAD 1185 bp DNA linear VRT 18 -APR-2005 Cairina moschata (duck) gene for alpha-D globin. X 01831. 1 GI: 62724 alpha-globin; globin. Cairina moschata (Muscovy duck) Cairina moschata Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Archosauria; Aves; Neognathae; Anseriformes; Anatidae; Cairina. 1 (bases 1 to 1185) Erbil, C. and Niessing, J. The primary structure of the duck alpha D-globin gene: an unusual 5' splice junction sequence EMBO J. 2 (8), 1339 -1343 (1983) 10872328 Data kindly reviewed (13 -NOV-1985) by J. Niessing.
Gen. Bank format - ORIGIN section ORIGIN 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 // ctgcgtggcc cagggtgcta agcctgccac gtgggagaag gctgggccca tgggctggga aaaactgact ttcccccact gcggctgccc ctcagcaacc gactagggtctgagtt gggtaccagg gtgggccaga gggggactca tccggagcag tggtgctggc agttcttgtc cccttgcacc gggcatcggg tcagcccctc taagagctcg gccgctgccg gtggcc gggggcactc cccagagcgc ggcctcgctc tcgacctgca tgggcaatgc tgcatgccta cttgggtctg tcctggggtc gtcctggggg ggctgggatt gggcctcagg gggtactaag cgcacacctg cgccgtggct ttcaataaag ggtcccaggg cacccctcca gccccgcggg ccatgctgac accaggagga acagggtggg cacggggtgc cggcaggatg tcccggctct cgtgaagagc caacctgcgt ggggtctgag tggcagtcct ccagcagcca gtgtttggaa gggactcggg ccctggtttg ggcaaagact gccgtgctgg acaccattac agggctgggt cgctgataag tgtctccacc cgccgaggac attcggaagt cagcagggag gggctgagat ttcctcgcct gaacaggtcc ctggacaacc gttgaccctg ggtgtggggt gggggctgag gacagcaggg tgggagctgg gggggactga ccttgcagct acagccccga ctgaaaagta cacagctctg tgcttccaca ataaggccag acagaaaccc aagaagctca gaagctctgc caggagccct gggcaaagca acccccagac gtggccatgg tcagccaggc tcaacttcaa gcagggtctg ggccagggtc gctgggattg gcaggggcta gggagactca gctggcacag gatgct cagatgagcc tgtctgtgtg catcc ggcgggagcg gtcagttgcc tcgtgcaggt agaggtgtgg gcagcgggtg gcagggcacc caagacctac caagaaagtg cctgtctgag ggcaagcggg ggggtccagg ctgtggtctt catctgggat gggccagggt gggccatctg tgcttccagg gcctttgaca actgca tgctgggact
Gen. Bank format - FEATURE section FEATURES source CAAT_signal TATA_signal precursor_RNA exon CDS repeat_region intron repeat_region exon intron exon poly. A_signal Location/Qualifiers 1. . 1185 /organism="Cairina moschata" /mol_type="genomic DNA" /db_xref="taxon: 8855" 20. . 24 69. . 73 101. . 1114 /note="primary transcript" 101. . 234 /number=1 join(143. . 234, 387. . 591, 939. . 1067) /codon_start=1 /product="alpha D-globin" /protein_id="CAA 25966. 2" /db_xref="GI: 4455876" /db_xref="GOA: P 02003" /db_xref="Inter. Pro: IPR 000971" /db_xref="Inter. Pro: IPR 002338" /db_xref="Inter. Pro: IPR 002340" /db_xref="Inter. Pro: IPR 009050" /db_xref="Uni. Prot/Swiss-Prot: P 02003" /translation="MLTAEDKKLIVQVWEKVAGHQEEFGSEALQRMFLAYPQTKTYFP HFDLHPGSEQVRGHGKKVAAALGNAVKSLDNLSQALSELSNLHAYNLRVDPVNFKLLA QCFQVVLAAHLGKDYSPEMHAAFDKFLSAVAAVLAEKYR" 227. . 246 /note="direct repeat 1" 235. . 386 /number=1 289. . 309 /note="direct repeat 1" 387. . 591 /number=2 592. . 939 /number=2 940. . 1114 /number=3 1095. . 1100 1114
Exercise: Gen. Bank • Work in groups of 2 -3 people. • The exercise guide is linked from the course programme. • Read the guide carefully - it contains a lot of information about Gen. Bank.
- Slides: 46