Mutations and Gel Electrophoresis

1. Mutations and Gel Electrophoresis

2. Mutations • “Changes in the DNA sequence that are inherited” • Can have a negative consequence, no consequence, or a positive consequence

3. Silent Mutations • Has no consequence (neither good nor bad) • Can happen in 2 ways: • A mutation occurs in an intron, which is spliced out during mRNA processing. • A mutation occurs in the DNA which does not change the amino acid

4. Silent Mutations - Example Example (No change in aa) 5’ – AUG AAG UUU GGC UAA – 3’ Met Lys Phe Gly Stop 5’ – AUG AAG UUU GGU UAA – 3’ Met Lys Phe Gly Stop Original (normal) mRNA Mutated (Base C changed to U) mRNA But no change in amino acid

5. Missense Mutations • A change in a nitrogenous base leads to a change in the amino acid produced. 5’ – AUG AAG UUU GGC UAA – 3’ Met Lys Phe Gly Stop 5’ – AUG AAG UUU AGC UAA – 3’ Met Lys Phe Ser Stop Original (normal) mRNA Missense mutation (G base changed to A) = A different amino acid

6. Nonsense Mutations • A change in a nitrogenous base leads to a premature stop codon 5’ – AUG AAG UUU GGC UAA – 3’ Met Lys Phe Gly Stop 5’ – AUG UAG UUU GGC UAA – 3’ Met Stop Original (normal) mRNA Nonsense mutation (A base changed to U) = Premature stop codon

7. Classification • Missense and Nonsense mutations arise because of a base pair substitution • In other words, the nitrogenous base itself may change, but the number of nitrogenous bases don’t change

8. Deletion Mutations • One or more bases are deleted 5’ – AUG AAG UUU GGC UAA – 3’ Met Lys PheGly Stop 5’ – AUG AAG UUG GCU AA – 3’ Met Lys LeuAla Original (normal) mRNA Deletion of the base “U” = The “reading frame” shifts, and new amino acids result

9. Insertion Mutations • The addition of one or more bases 5’ – AUG AAG UUU GGC UAA – 3’ Met Lys Phe Gly Stop 5’ – AUG UAA GUU UGG CUA – 3’ Met Stop Original (normal) mRNA Insertion of the base “U” = The “reading frame” shifts, and a stop codon results. This frameshift insertion cause a nonsense mutation

10. Classification • Frameshift mutations occur because of insertions or deletions • All of these mutations (missense, nonsense, insertions, deletions) are called point mutations meaning that they occur only with one base pair

11. Classification • Another category of mutations involves large segments of DNA • These are called chromosomal mutations TRANSLOCATIONS INVERSIONS

12. How do mutations arise? • What causes mutations? • List examples • Spontaneously • Mutagenic agents (chemicals that cause mutations, e.g. X rays, UV radiation, cosmic rays, chemicals)

13. Try It! The following strand of mRNA represents the “normal” (aka wild type) strand. 5’ – AUG GGG UUU AUC CUA UAG – 3’ This strand is hit by UV radiation and turns into: 5’ – AUG GGG UUG AUC CUA UAG – 3’ • Write the amino acid sequences for both strands • What kind of mutation occurred? Be very specific. • What would happen if the “UUG” changed into “UAG”?

14. Gel Electrophoresis Prep for Tomorrow’s Lab

15. Gel Electrophoresis • Is a procedure used to separate DNA fragments

16. Gel Electrophoresis – General Steps • Prepare the DNA samples by adding restriction enzymes to them. Restriction enzymes will cut the DNA into smaller fragments. • Add loading dye to your DNA samples. This will allow you to see the DNA as it migrates down the gel. • Pour the agarose gel (with buffer) into your electrophoresis tray.

17. Gel Electrophoresis – Steps Cont’d 4. Load your DNA into the “wells” using a micropipette. Make sure you write down which sample you are putting into which well (e.g. Well 1 = Crime Scene Suspect, Well 2 = DNA Sample 1, Well 3 = DNA Sample 2, etc.) 5. Plug in your electrodes, and turn on the power to 100V for 30 minutes. 6. Your DNA fragments will separate based on size.

18. Gel Electrophoresis • Remember that this lab is informal • Try to answer the questions as you perform the lab, it will give you less homework to do afterwards