1. MCB 130L
Lecture 1: DNA

2. Central Dogma of Molecular Biology
This week: Molecular Biology Module
Proposed by Francis Crick in 1958
Oversimplification b/c RNA can RT to DNA; RNA can replicate itself, noncoding RNAs, alternative splicing, introns, etc.
Molecular biology is the study of the process of replication, transcription and translation of the genetic material
Interesting fact: Crick did not have a PhD at the time of his discovery, switched from physics to mol biol after a WWII bomb destroyed his lab equipment.
Proposed by Francis Crick, 1958

3. Recombinant DNA technology
Creation of a novel combination (i.e. human and bacteria DNA)
Applications:
Cloning
Sequencing
Modification
Mutagenesis
Creation of novel fusion genes
Cloning=making multiple copies of a fragment of DNA, i.e. gene
Sequencing—If you’re interested in obtaining a DNA sequence, you can use recombinant DNA technology (i.e. PCR, cDNA/DNA library) to sequence that region
Modification—mutagenesis: function of gene, necessity of gene fragment, amino acid, etc.
fusion genes: improving activity of protein i.e. Maxygen gene shuffling technology
ease of purification, fluorescent tagging, etc.
cDNA, RNAi libraries

4. Importance of recombinant DNA
Basic research
Gene structure
splicing
transcriptional regulation
Protein function
domain structure
post-translational modifications
phosphorylation sites
Biotechnology
Insulin, growth hormone
Gene shuffling
Gene therapy
Gene structure—including transcriptional regulation
Protein function—domains, phosphorylation/post-translational modifications, etc.

5. Experimental Proposal: To determine the role of HexB in immune defense against tuberculosis?
Recombinant DNA technology
Clone HexB
Recombinant DNA technology
Clone HexB
Mutate HexB
Knockout/overexpress HexB
Purify Protein
Biochemical assays
Protein-protein interaction
X-ray crystallography
Antibody production
phenotype
domain characterization
phenotype
cellular localization

6. Essential steps in the generation of recombinant DNA
DNA (genomic, plasmid, PCR, ….)
DNA fragmentation/digestion
DNA Separation and purification
Forming recombinant DNA: ligation
Cloning DNA: Transformation, selection and amplification
Plasmid=best example b/c can be amplified in bacteria
E. Coli replication=20 minutes
Can obtain huge amounts of DNA of interest
Transition: PCR to amplify fragment of interest, then insert into plasmid to make recombinant DNA

7. Amplification of specific DNA sequences:
Polymerase Chain Reaction (PCR)
Applications:
1. general amplification
2. diagnostics
3. isolating DNA from ancient organisms
4. forensics
Amplification for cloning, sequencing, basic research purposes
Diagnostics for genetic diseases, infectious diseases, i.e. XDR TB
Isolating DNA—Jurassic Park
Forensics—identifying suspects/victims of criminal cases
Invented by Kary Mullis (UCB PhD) while at Cetus Corp., Emeryville
1993 Nobel Prize in Chemistry

8. PCR movie

9. Amplification of specific DNA sequences:
Polymerase Chain Reaction (PCR)
1. Logarithmic amplification: # of copies = 2n, n = # of cycles
2. Sensitive: a single molecule can be amplified
3. Contamination a problem!
Contamination/sensitivity issues: especially when doing diagnostics, quantitative pcr, etc.

10. Amplification of specific DNA sequences:
Polymerase Chain Reaction (PCR)
1. Taq DNA polymerase from thermophilic bacteria
(Thermus aquaticus, error rate 1/105)
2. dNTPs (dATP, dCTP, dTTP, dGTP)
3. Template = DNA to be amplified
4. Primers: nucleotides complementary to template
5. Temperature cycling: cycles
Denaturation 95ºC
Annealing 55ºC to 60ºC
Extension 72ºC
Taq withstands denaturing temperatures
Pfu polymerase=proofreading

11. Amplification of specific DNA sequences:
Polymerase Chain Reaction (PCR) 5’ 3’
72ºC
(Polymerase optimal
temperature) 5’ 3’
95ºC
(Denaturation)
55ºC
(Annealing) 3’ 5’
Cycle 1 (same procedure will be repeated times)

12. Essential steps in the generation of recombinant DNA
DNA (genomic, plasmid, PCR, ….)
DNA fragmentation/digestion
DNA Separation and purification
Forming recombinant DNA: ligation
Cloning DNA: Transformation, selection and amplification
Plasmid=best example b/c can be amplified in bacteria
E. Coli replication=20 minutes
Can obtain huge amounts of DNA of interest
Transition: PCR to amplify fragment of interest, then insert into plasmid to make recombinant DNA

13. Cloning movie

14. Cloning DNA: plasmid vectors
Origin of replication
Polylinker or multiple cloning site (MCS)
Ampr gene (selectable)
What would happen if any of these 3 features were missing?
MCS: Nowhere to add the DNA of interest
(Bacteriophages = alternative cloning vector)

15. Multiple cloning site
Region of plasmid containing multiple restriction enzyme sites to enable insertion of DNA of interest

16. Cutting DNA: restriction enzymes
Site specific endonucleases produced by bacteria
Recognize palindromic sequences (same 5’ --> 3’ on both strands)
Evolved to cleave bacteriophage DNA

17. Cutting DNA: restriction enzymes
How do bacteria survive with restriction enzyme that cleaves DNA?
- bacteria DNA is protected from cleavage by methylation
Figure 4: Bacteria cells that produce restriction endonucleases also produce modification enzymes that methylate bases in the recognition site.

18. Separating and purifying DNA fragments: gel electrophoresis
DNA is negatively charged
Moves to the (+) pole in electric field
Ethidium bromide intercalates DNA, fluoresces in UV light

19. Essential steps in the generation of recombinant DNA
DNA (genomic, plasmid, PCR, ….)
DNA fragmentation/digestion
DNA Separation and purification
Forming recombinant DNA: ligation
Cloning DNA: Transformation, selection and amplification
Plasmid=best example b/c can be amplified in bacteria
E. Coli replication=20 minutes
Can obtain huge amounts of DNA of interest
Transition: PCR to amplify fragment of interest, then insert into plasmid to make recombinant DNA

20. Forming recombinant DNA molecules: ligation
- T4 DNA ligase
Requires ATP
Phosphodiester bond
Ligation of sticky ends is
more efficient than blunt
Ligation—creates phosphodiester bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another

21. Cloning DNA molecules: transformation, selection and amplification
Transformation = Introduction of plasmid into bacteria
Make “competent” bacteria
Add DNA
Inefficient uptake
Selection for antibiotic resistance
Amplification: Bacteria replicate w/ plasmid

22. Other Methods in recombinant DNA technology
Southern blot
DNA sequencing

23. Southern Blot
Microarray technology evolved from Southern blotting

24. DNA Sequencing: dye terminator sequencing
Dideoxynucleotide triphosphates/chain terminators
Dye-terminator sequencing, capillary electrophoresis

25. This week’s lab:
PCR
Restriction Digests
Agarose Gel Electrophoresis