Lab 2: Biochemical and Gene Techniques - DNA and Protein Sequence Databases

DNA and Protein Sequence Online Databases

• There are two types of sequence databases: primary and secondary databases
• Primary databases contain experimental results in an accessible format but are not consensus sequences
• Secondary databases are curated to reflect consensus sequences from multiple experiments and use primary databases as their sources
• Examples of primary databases: DDBJ (DNA Databank of Japan), EMBL (European Molecular Biology Laboratory), and GenBank
• Abbreviations: NCBI (National Center for Biotechnology Information), BLAST (Basic Local Alignment Search Tool)

Entrez and Sequence Search

• Entrez is a data retrieval system developed by NCBI that provides integrated access to a wide range of data domains
• Search goals:
  • Identify a representative, well-annotated mRNA or protein sequence record
  • Retrieve associated literature
  • Identify conserved domains within the protein
  • Identify similar proteins
  • Find a resolved three-dimensional structure for the protein or identify structures with homologous sequence
• Steps to start: Go to the NCBI website, select a database (nucleotide or protein), and search for a gene or protein of interest

BLAST Introduction

• BLAST (Basic Local Alignment Search Tool) is a sequence comparison algorithm optimized for speed and sensitivity
• Selecting a BLAST program: nucleotide blast (blastn), protein blast (blastp), blastx, tblastn, and tblastx
• BLAST programs compare a query sequence against a database or vice versa

BLAST Results

• The blastp results page shows around 100 "Hits", or other protein sequences showing at least some similarity to the query sequence
• The illustration with red bars is a diagrammatic representation of how the query sequence lines up with other sequences in the database along the primary structure of the protein
• The two scores at the right (Ident and E value) indicate the degree of similarity

Clustal Omega and Multiple Sequence Alignment

• Clustal Omega is a DNA and protein multiple sequence alignment tool
• Multiple alignments of protein sequences are important tools in studying sequences and provide identification of conserved sequence regions

Glossary

• Alignment: The process of lining up two or more sequences to achieve maximal levels of identity and conservation
• Algorithm: A fixed procedure embodied in a computer program
• Bioinformatics: The merger of biotechnology and information technology with the goal of revealing new insights and principles in biology
• Conservation: Changes at a specific position of an amino acid or DNA sequence that preserve the physico-chemical properties of the original residue
• Domain: A discrete portion of a protein assumed to fold independently of the rest of the protein and possessing its own function
• Gap: A space introduced into an alignment to compensate for insertions and deletions in one sequence relative to another
• Global Alignment: The alignment of two nucleic acid or protein sequences over their entire length
• Identity: The extent to which two nucleotide or amino acid sequences are invariant
• Local Alignment: The alignment of some portion of two nucleic acid or protein sequences
• Motif: A short conserved region in a protein sequence
• Proteomics: The systematic analysis of protein expression in normal and diseased tissues that involves the separation, identification, and characterization of all of the proteins in an organism
• Query: The input sequence (or other type of search term) with which all of the entries in a database are to be compared