DNA as Biological Information Rasmus Wernersson Henrik Nielsen

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

Hvad er gener? rough strain & DNA from killed smooth strain

DNA: sammensætning • Omkring 1950 vidste man at DNA var en polymer af nukleotider – nærmere bestemt deoxyribonukleotider. • De fire nukleotider udgør DNA er kun forskellige i deres nitrogen-base. • Der er to puriner (adenin og guanin) og to pyrimidiner (cytosin og thymin). Uracil (en pyrimidin) forekommer kun i RNA

Deoxyribonukleotider 5’ 4’ 1’ 2’ 3’ deoxy

DNA: sammensætning 2 Chargaff’s regel: Der er lige mængder A og T samt lige mængder C og G. (mens forholdet mellem G+C og A+T kan variere)

DNA: Røntgenkrystallografi: Rosalind Franklin DNA præparation: Maurice Wilkins Modelbygning og tolkning af røntgenspektre: Francis Crick & James Watson

Watson & Crick 1953

DNA Struktur

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’ AGACC 3’ 3’ TCTGG 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 separated using gel electrophoresis – Typically 1% agarose – 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 happened 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 international DNA databases. • Gen. Bank is hosted by NCBI: National Center for Biotechnology Information. • Gen. Bank has existed 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.