DNA Sequence Analysis

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DNA Sequence Analysis. Broad and Long Term Objective. To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis. Research Plan. Preparation of Competent Cells and Bacterial Transformation. Growth of Transformant and Plasmid MiniPrep. Cycle Sequencing. - PowerPoint PPT Presentation

Text of DNA Sequence Analysis

  • DNA Sequence Analysis

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

  • Research PlanPreparation of Competent Cells and Bacterial TransformationGrowth of Transformant and Plasmid MiniPrepCycle SequencingSequence analysis

  • Todays Laboratory Objectives

    To learn how to characterize a DNA sequence using various web based bioinformatics tools including:1. BLASTN- has this piece of DNA been sequenced before? Does it look like anything already in GeneBank at the nucleotide level?2. BLASTX- Can we identify the putative function of the transcripts?3. ORF Finder- What does the open reading frame look like? Do we have a full length clone with an identifiable start and stop codon?4. ClustalW- How does it compare with other sequences either at the nucleotide or amino acid level? What residues are conserved and thus likely to be important? And what residues are divergent?

  • BLAST Database Search ToolBLAST (Basic Local Sequence Alignment Tool)Available on the internet and downloadableQuick and simplehttp://www.ncbi.nlm.nih.gov/

  • The BLAST Family

    ProgramQuery SequenceDatabase TargetBLASTNNucleotide (both strnds)Optimized for speed not accuracyNot good for distant homologuesDust Option (low complexity)Nucleotide DatabaseBLASTXNucleotide translated 6 framesLess sensitive to sequence errors and mismatchesUseful for preliminary data/ESTDust Filter OptionProtein DatabaseTBLASTXNucleotide translated 6 framesGood for ESTs and Single Pass Sequences, Very SlowNucleotide DatabaseTranslated 6 framesBLASTPProteinProtein DatabaseTBLASTNProteinProteins against nucleotides and ESTsNucleotide DatabaseTranslated 6 frames

  • The Blast AlgorithmIdentify HSPs (High Scoring Segment Pairs)default 11 bp or 3 aaPerfect match

    Slide query and target sequence across each other until the maximum number of HSP for that target is found

  • The Blast AlgorithmScore the Alignment a scoring matrx such as BLOSUM62 or PAM is used gaps introduced between GSPs during sliding get negative score a match gets a positive scoretotal alignment score is subjected to statistical analysis to calculate the significance vs. chance of the scoreRepeat for every sequence in the target databaseReturn total results

  • Paste Sequence hereSubmit Search by Clicking Here

  • Execute Search by Clicking Format

  • BLASTX Results

  • Interpreting BLAST ResultsLengthE-ValueBit ScoreIdentitiesPositives

  • Begin with ATG start codonEnd with TAA, TAG, or TGA stop codonsCan occur in any six possible reading frames

    Sense Strand: Frame +1 Frame +2 Frame +3Antisense Strand: Frame -1 Frame 2Frame -3NCBIs ORF FINDER and Open Reading Frames

  • ORF Finder AlgorithmIterates over all frames:Iterate to the end of frameFind first/next Start codonContinues to the next Stop codonRecords the size and location of ORFList OFRs sorted by length in descending order

  • www.ncbi.nlm.nih.gov/gorf/gorf.html

  • Graphical ViewORF TableMinimum ORF Length: Can Redraw with lower cut-offClickable

  • Submit for BLASTSelected ORFORF LengthORF Translation

  • Multiple Sequence Alignment with Clustal WHomologous residues in a set of sequences are aligned together in columnsIdeally, homology reflects structural and evolutionary conservationEvolutionary history of a residue can be deduced from sequence alignments of sequences from different organisms

  • http://www.ebi.ac.uk/clustalw/

  • Alignment EditorPairwise Scores

  • Download fileColored Alignment