1. Sequence Analysis

2. DNA and Protein sequences are biological information that are well suited for computer analysis Fundamental Axiom: homologous sequences share an evolutionary ancestor and are almost surely performing the same or a similar function

3. Sequence Analysis topics for today Restriction enzyme sites for diagnostics and cloning Open reading frame analysis Conceptual translation Oligo primer design Sequence alignments

4. Sequence Analysis Alignments document homologous relationships DNA sequence alignments - best for showing identity Protein sequence alignments best for showing similarity

5. Types of Alignments

6. In Class Tutorial Introduction to File Formats –Examples of file formats –Utilities to change formats Restriction Analysis –Web tools for restriction analysis –Local programs

7. In Class Tutorial Open reading frame analysis Reverse complement Capturing output to an MS Word doc Oligo Primer Design for PCR and sequencing Alignments –global and local

8. Sequence File Formats FASTA –Simplest format –Easy to create by hand on a word processor

9. FASTA First line must start with > followed by seq name Second line to end = sequence No numbers or spaces Seq can be UPPER or lower case

10. File Formats Some sequence analysis program take input sequences in FASTA format ONLY ReadSeq is a web based utility that converts many file formats to FASTA More and more programs will accept multiple file formats as input

11. Mono-Space Fonts Every character uses the same space = mono space ATG and C use the same space on a line W and. use the same space on a line Critical for sequence alignments to stay aligned

12. Mono-Space Fonts NOT a Monospace font

13. Primer Design Primers are chemically synthesized oligonucleotides Used for sequencing and PCR Bad primer design can result in reaction failures

14. Primer Design Matters

15. Primer Design TM 55-60°: PCR primer pairs need to have similar TM’s GC content 40-60% (Biased to 5’ end) Length = 17-25nt Low self complementarity (Palindromes) < 3/5 3’ bases G/C (no GC clamp at 3’ end) Low complementarity between primers (avoid primer dimer) Blast search primers – avoid repetitive DNA Small amplicon size increases PCR efficiency Avoid runs of one base

16. Primer Design: GC Clamps cause false priming