Mutations

Missense Point Mutations Neutral Synonymous/Silent : Base change that does NOT change the amino acid coded Ex. AGG → CGG both Arg Non-Synonymous - Conserved: Base change results in a different but similar amino acid Same charge and shape Ex. AAA → AGA Lys to Arg both basic amino acids

Missense Point Mutations Non-Synonymous-Semi conserved: Base change resulting in a different but similar amino acid Same shape but different charge Ex. CGC → CUC Arg (Polar) to Leu (Non-polar) Non-Synonymous - Non conserved Base change resulting in totally different amino acids Different shape different charge

Point Mutations Nonsense point mutation: Base change resulting in the creation of a premature stop codon within the ORF Causes premature translation termination Truncated protein Indel – Insertion or deletion of a single base within the ORF Changes reading frame Changes protein sequence May cause premature termination

Homologues Nucleotide homologues Protein homologues DNA sequences with greater than 70% identity Protein homologues Protein sequences with greater than 25% identity

Homologues Orthologues : Paralogues : Homologues found in different organisms which have a common ancestor Duplication followed by speciation Paralogues : Homologues found within the same species Duplication prior to speciation

Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis(SDS-PAGE)

Introduction Standard test used to determine presence and molecular weight of proteins Widely used in biochemistry, forensics, genetics and molecular biology Laemmli system of SDS-PAGE was first introduced in 1970s

Principle Separates proteins in an electric field Migrate through a semisolid medium from anode to cathode terminal Proteins migrate at different rates according to molecular size of proteins

Sds-coated large proteins migrate slowly through the gel matrix and small proteins migrate quickly through the matrix The nearer the band to the well, the larger the molecular size of protein

What is SDS? Negatively charged detergent sodium dodecylsulfate (SDS) Used to denature and linearize the proteins Confers a uniform negative charge to proteins

What is PAGE? Polyacrylamide is used to form a gel, a matrix of pores which allow molecules to migrate at different rates Pore size is determined by the concentration of acrylamide The higher the concentration, the smaller the size of pores

Visualization of protein bands Coomassie Blue staining Traditional method requires staining followed by destaining to remove background gel staining Most common and least sensitive Limited to ~100ng of protein

Applications Determine purity of protein samples Determine molecular weight of protein Quantifying proteins Blotting applications

Final exam – 3 hours 10 bioinfo questions – 10 points 5 calculations – 10 points 5 theoretical questions – 5 points 2 out of 3 problems – 20 points each 5 parts/problem Bonus problem – 5 points

Problems Problem 1 Problem 2 Transcription/Translation Northern analysis Protein expression Problem 2 DNA markers RFLPs and VNTRs

Problems (cont’d) Problem 3 Bonus Problem Tm and stringency Hybridization Cloning Ligations and transformations Bonus Problem Southern analysis Restriction mapping Restriction enzymes