MCB 130L Lecture 1 1. How to get the most from your time in lab 2. Recombinant DNA 3. Tips on giving...
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Transcript of MCB 130L Lecture 1 1. How to get the most from your time in lab 2. Recombinant DNA 3. Tips on giving...
MCB 130L Lecture 1
1. How to get the most from your time in lab
2. Recombinant DNA
3. Tips on giving a Powerpoint talk
1. How to get the most from your time in lab
1. Be well prepared: know what you are doing and why
2. Be organized
3. Be systematic in your work
4. Take careful and thoughtful notes
5. Clean up after yourself when done!!
2. Recombinant DNA technology
Recombinant DNA: Creation of a novel combination (e.g., human and bacteria DNA)
Applications: 1. Cloning = obtaining multiple copies 2. Sequencing 3. Modification: Mutagenesis Creation of novel fusion genes
…………
Importance
• Biotechnology (e.g., insulin, growth hormone)
• Basic research (gene structure, function, conservation)
• Gene therapy
Which genomes have been sequenced?
• Viruses
• Phage
• Organelle genomes
• Plants
• Model Organisms: yeasts, flies, worms
• Vertebrates (including humans)
1. DNA (genomic fragment, plasmid, PCR, ….
1. DNA fragmentation/digestion
2. DNA Separation and purification
3. Forming recombinant DNA: ligation
4. Cloning DNA: Transformation,selection and amplification
Essential steps in the generation of recombinant DNA
Cloning DNA: plasmid vectors
Origin of replication
Ampr gene (selectable)
Polylinker or multiple cloning site (MCS)
Cutting DNA: restriction enzymes
Site specific endonucleases produced by bacteriaRecognize palindromic sequences (same 5’ --> 3’ on both strands)Evolved to cleave bacteriophage (viral) DNA
blunt ends
Sticky ends:5’ overhang
5’ overhang
3’ overhang
Cutting DNA: restriction enzymes
Cutting DNA: restriction enzymes
1.Numerous restriction enzymes2.Most cleave at a unique sequence3.Named for bacterial species
Figure 4: Bacteria cells that produce restriction endonucleases also produce modification enzymes that methylate bases in the recognition site.
Cutting DNA: restriction enzymes
How do bacteria survive with restriction enzyme that cleaves DNA?- Restriction sites in bacteria DNA are protected from cleavage by methylation
Separating and purifying DNA fragments: gel electrophoresis
DNA is negatively chargedMoves to the (+) pole in electric field
Separating and purifying DNA fragments: gel electrophoresis
Ethidium bromide - intercalates between base pairs - Fluorescent when illuminated
with UV light
Danger: Mutagen - UV light
Forming recombinant DNA molecules: ligation
Involves ligase:- T4 (bacteriophage ) ligase
- Needs ATP , 5’ phosphate
-Ligation of sticky ends is more efficient than blunt
Cloning DNA molecules: transformation, selection and amplification
1. Transformation = Introduction of plasmid into bacteria- Treat bacteria with CaCl2 to make
them competent- Add DNA- Uptake inefficient
2. Selection for Ampicillin
3. Amplification: Bacteria replicate plasmid
Amplification of specific DNA sequences:Polymerase Chain Reaction (PCR)
Applications:
1. general amplification 2. diagnostics 3. isolating DNA from ancient organisms 4. forensics….
Invented by Kerry Mullis, UCB PhD, while at Cetus1993 Nobel Prize in Chemistry
Amplification of specific DNA sequences:Polymerase Chain Reaction (PCR)
1. Logarithmic amplification: # of copies = 2n, n= # of cycles2. Sensitive: a single molecule can be amplified3. Contamination a problem!
Amplification of specific DNA sequences:Polymerase Chain Reaction (PCR)
Technique uses:
1. DNA polymerase from thermophilic bacteria ex: Taq from thermus aquaticus (no proofreading, error rate 1/105)
2. dNTPs (dATP, dCTP, dTTP, dGTP)3. Template = DNA to be amplified4. primers: 18-20 nucleotides complementary to template5. Temperature cycling: 20-30 cycles
95ºC: denaturation55ºC to 60ºC annealing72ºC Extension
Amplification of specific DNA sequences:Polymerase Chain Reaction (PCR)
5’ 3’
3’ 5’
5’ 3’ 5’ 3’ 5’ 3’
3’ 5’3’ 5’3’ 5’
95ºC(Denaturation)
72ºC(Polymerase optimaltemperature)
55ºC(Annealing)
Cycle 1 (same procedure will be repeated 25-30 times)
Amplification of specific DNA sequences:Polymerase Chain Reaction (PCR)