1. Review of Laboratory 3 Spectrophotometric determination of DNA quantity, purity Abs 260 nmAbs 280 nmAbs 320 nmAbs 260/Abs 280 0.05241 0.03110 0.00261 1.75 Restriction digest, agarose gel electrophoresis Expected results:

2. Genomic DNA Uncut Plasmid DNA Lab 3 electrophoresis results WR problems with ladder (λ HindIII) incomplete digestion what do these banding patterns represent?

3. Our good friend Phaeodactylum tricornutum

4. Cycle Sequencing and Bioinformatics Tools

5. Broad and Long Term Objective To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis

6. Research Plan Preparation of Competent Cells and Bacterial Transformation Growth of Transformant and Plasmid MiniPrep Cycle Sequencing Sequence analysis

7. Today’s Laboratory Objectives 1.To understand the theoretical basis of Sanger- type DNA sequencing 2.To learn how to analyze your E. huxleyi cDNA sequence using various web-based bioinformatics tools

8. What is Cycle Sequencing? Based on the Sanger Dideoxy chain termination method Based on the Sanger Dideoxy chain termination method(1974) DNA synthesis reaction whereby fluorescently labeled dideoxynucleotides are incorportated into the newly replicated DNA by DNA polymerase in a primer extension reaction DNA synthesis reaction whereby fluorescently labeled dideoxynucleotides are incorportated into the newly replicated DNA by DNA polymerase in a primer extension reaction Separation of labeled fragments by polyacrylamide gel electrophoresis Separation of labeled fragments by polyacrylamide gel electrophoresis

9. * Mix of dNTPs and flourescently labeled ddNTPs ddCTP = blue, ddGTP = yellow, ddATP = green, ddTTP = red DNA sequencing: ddNTPs Incapable of phosphodiester bond formation

10. DNA sequencing: DNA synthesis and chain termination dsDNA template single primer chain termination by incorporation of ddNTP Collection of labeled DNA fragments differing In length by 1 base pair Amplitaq polymerase 96º C 50º C 60º C

11. Labeled DNA fragments

12. Reaction Products are Separated on a Polyacrylamide Gel 5’-ATGATA…………………-3’ DNA fragments run on a 4-8% acrylamide gel allowing visualization of 1 bp size differences At the bottom of the gel, a stationary laser excites the passing fluorescent tags on the ddNTPs and fluorescence is measured by a detector software programs (e.g. phred) convert fluorescence readings to sequence

13. DNA sequence analysis ORF Finder- http://www.ncbi.nlm.nih.gov/gorf/*http://www.ncbi.nlm.nih.gov/gorf/ BLASTN- http://www.ncbi.nlm.nih.gov/BLAST/*http://www.ncbi.nlm.nih.gov/BLAST/ BLASTX- http://www.ncbi.nlm.nih.gov/BLAST/*http://www.ncbi.nlm.nih.gov/BLAST/ Clustal W- http://www.ebi.ac.uk/clustalw/*http://www.ebi.ac.uk/clustalw/ For more information- http://www.ncbi.nlm.nih.gov/Education/index.html http://www.ncbi.nlm.nih.gov/Education/index.html

14. ORF finder ORF (Open reading frame) finder: http://www.ncbi.nlm.nih.gov/gorf/http://www.ncbi.nlm.nih.gov/gorf/ Why would I use it? Is there a potential protein-coding region in this sequence? Is the ORF complete (with defined start and stop codons)? Identifies ORFs in DNA sequences How does it work? Scans all six possible reading frames (+1, +2, +3, -1, -2, -3) for potential start codons (ATG) followed by in-frame stop codons (TAA, TGA, TAG) Assumption: larger ORFs more likely to code for proteins Useful for prokaryotic genomic DNA/cDNA, eukaryotic cDNA

16. ORF table Clickable

17. Selected ORF BLAST selected ORF ORF length Translation

18. BLAST Database Search Tool BLAST- Basic Local Alignment Search Tool: http://www.ncbi.nlm.nih.gov/BLAST/ Why would I use it? Find similarity between your nucleotide/protein sequence and other seqeuences deposited in very large (updated daily, >25 million entry) public databases Assumption 1: Similar DNA/protein sequences indicate descent from a common ancestral sequence (i.e. homology) Assumption 2: Similar protein sequences indicate a similar structure and function

19. ProgramQuery SequenceDatabase Target BLASTNNucleotide (both strands) Optimized for speed not accuracy Not good for distant homologs Dust Filter Option (low complexity) Nucleotide Database BLASTXNucleotide translated 6 frames Less sensitive to sequence errors and mismatches Useful for preliminary data/EST Dust Filter Option Protein Database BLASTPProteinProtein Database The BLAST Family

20. How does it work? The BLAST Algorithm Performs similarity search between your query sequence and a nucleotide/protein database. Pairwise alignment (two sequences compared) Query sequence is split into small fragments (words) of default sizes of 11 bp or 3 amino acids Words are compared to every sequence in the target database (sliding window) ATTGTACCGTA …GCGAT-ATACAGTTTTA…

21. How does it work? The BLAST Algorithm Every window is scored using a scoring matrix, e.g. Every window is scored using a scoring matrix, e.g. BLOSUM62 or PAM BLOSUM62 or PAM Nucleotide matrix- Match = +3, Substitution = -1, Gap = -5 Nucleotide matrix- Match = +3, Substitution = -1, Gap = -5 Amino acid matrix- more complex relationships between Amino acid matrix- more complex relationships between amino acid substitutions amino acid substitutions Goal to identify HSP’s (High Scoring Segment Pairs), Goal to identify HSP’s (High Scoring Segment Pairs), examine adjacent regions of sequence (Local alignment) examine adjacent regions of sequence (Local alignment) total alignment score is subjected to statistical analysis to total alignment score is subjected to statistical analysis to calculate the significance vs. random chance of the score calculate the significance vs. random chance of the score ATTGTACCGTA …GCGAT-ATACAGTTTTA…

24. BLASTX Results

25. Interpreting BLAST Results Length E-Value (<1 x 10 -3 ) Bit Score (>30) Identity (>25% amino acid i.d.) Positives

26. Clustal W: multiple sequence alignments ClustalW: http://www.ebi.ac.uk/clustalw/ http://www.ebi.ac.uk/clustalw/ Allows comparison of more than two sequences, with identical and similar residues aligned in columns Allows comparison of more than two sequences, with identical and similar residues aligned in columns Assumption 1: similar sequences are structurally, functionally, and evolutionarily related Assumption 1: similar sequences are structurally, functionally, and evolutionarily related Assumption 2: specific conserved residues (a.a. or nt) are functionally important Assumption 2: specific conserved residues (a.a. or nt) are functionally important

27. Why would I do a multiple sequence alignment? Phylogenetic comparisons (more similar sequences are more closely related) Phylogenetic comparisons (more similar sequences are more closely related) Identify highly conserved amino acid/nucleotide sequences (may be critical for function) Identify highly conserved amino acid/nucleotide sequences (may be critical for function) Characterize gene family Characterize gene family Identify conserved regions for the design of PCR primers Identify conserved regions for the design of PCR primers

28. How does Clustal W work? True multiple sequence alignment is extremely computationally taxing True multiple sequence alignment is extremely computationally taxing Clustal W strategy: progressive pairwise alignments Clustal W strategy: progressive pairwise alignments A+B C+D E+F Consensus 1 Consensus 2 Consensus 3 + Consensus 4 + Final alignment

30. Pairwise scores Alignment editor

31. Colored alignment Download file