1.  Test Review Lecturer: David Mendez

2. But first  Take out a sheet of paper

3. Pop Quiz 1. Why do we increase the temperature for the first step of a PCR reaction? 2. What 3 things make up a dNTP? 3. You have this sequence of DNA: 5’AGCTTTACT3’. Your primers are 3 bases long. What are your primers? 4. State one difference between the leading strand and lagging strand. 5. Gel electrophoresis separates DNA based on what?

4. DNA  Know the structure of DNA  Bases, backbone, bonds  Central Dogma of Biology  DNA replication process  Leading v. lagging strand  Key players of replication

5. RNA  RNA structure  Bases, sugar  Different types of RNA  Transcription  Steps of transcription  The purpose of splicing  5’ cap and poly A tail

6. Proteins  What they’re made up of  Codons  Translation  mRNA  tRNA (anticodon)  Ribosomes

7. Mutations  Frameshift – changes in reading frame  Missense – substituting one amino acid for another  Nonsense – changing an amino acid so you now have a STOP codon  Silent – substitution that results in NO change in amino acid

8. Restriction Enzymes  Definition of a restriction enzyme  Recognition sequences  Palindrome sequence  Nucleases  Difference between blunt and sticky ends  Plasmid  Definition  Functions  Used as vectors

9. Palindrome Sequences

10. Restriction Enzymes Important Terms Nucleases: Enzymes that cleave DNA (ex: restriction enzymes) Endonuclease Exonuclease Basic Types

11. Practice: Blunt or Sticky? BLUNT STICKY

12. Plasmid A small, circular piece of DNA, separate from the chromosomal DNA. Replicates independently of the chromosomal DNA Usually encode genes that help the organism respond to environmental challenges Found in some yeast/fungi and bacteria Plasmid

13. Encode genes that confer resistance to antibiotics Enable conjugation - horizontal gene transfer – a way to take up exogenous DNA Plasmid Typical functions

14. Used as vectors for genetic engineering Vector = agent that transfers genetic material from one cell to another. –Replicate easily inside host bacteria –Readily accept and transfer new genes Plasmid Practical Applications

15. PCR  Definition of PCR  Items required for PCR and their function in PCR  The 3 steps of PCR and what’s occurring  Gel electrophoresis  How DNA travels  Ladder, positive control, negative control

16. PCR is a technique used to AMPLIFY a few copies of DNA exponentially. What is PCR???

17. How does it work?  You need 5 things:  Template DNA  Primers  Thermal cycling – increase and decrease the temperature  taq DNA polymerase  dNTPs

18. Overview  This cycle of heating and cooling is repeated many times to amplify the amount of DNA.

19. Gel Electrophores is  Run product on an agarose gel  Electric current is run through the gel to separate product based on size  Ensures the correct product was amplified

20. Gel electrophoresis  Ladder – DNA solution of varying sizes and used to compare samples  Positive control – a group where you expect an outcome  Negative control – a group where you expect nothing to happen  Larger fragments are closer to the top

21. Lab today  Simulation of restriction enzymes  Cut DNA representations and cut at specific sites (restriction enzyme)  You will be simulating 3 enzymes: EcoRI, BamHI, HindIII  2 activities using restriction enzymes