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)