1. Mutations

2. 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

3. 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

4. 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

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

6. 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

7. Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis(SDS-PAGE)

8. 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

9. 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

10. 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

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

12. 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

13. 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

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

16. 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

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

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