MCB 130L

Lecture 1: DNA

Central Dogma of Molecular Biology

Proposed by Francis Crick, 1958

Recombinant DNA technology

Recombinant DNA:

Creation of a novel combination (i.e. human and bacteria DNA)

Applications:

1. Cloning

2. Sequencing

3. Modification

Mutagenesis

Creation of novel fusion genes

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

Experimental Proposal:To determine the role of HexB in immune defense against tuberculosis?

Recombinant DNA technology

Clone HexB

Biochemical assays

Protein-protein interaction

X-ray crystallography

Antibody production

phenotype

cellular localization

Recombinant DNA technology

Clone HexB

phenotype

domain characterization

Purify ProteinMutate HexB Knockout/overexpress HexB

1. DNA (genomic, plasmid, PCR, ….)

2. DNA fragmentation/digestion

3. DNA Separation and purification

4. Forming recombinant DNA: ligation

5. Cloning DNA: Transformation,

selection and amplification

Essential steps in the generation of recombinant DNA

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 Kary Mullis (UCB PhD) while at Cetus Corp., Emeryville1993 Nobel Prize in Chemistry

PCR movie

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!

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: 18-20 nucleotides complementary to template

5. Temperature cycling: 20-30 cycles

Denaturation 95ºC Annealing 55ºC to 60ºC

Extension 72ºC

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 20-30 times)

1. DNA (genomic, plasmid, PCR, ….)

2. DNA fragmentation/digestion

3. DNA Separation and purification

4. Forming recombinant DNA: ligation

5. Cloning DNA: Transformation,

selection and amplification

Essential steps in the generation of recombinant DNA

Cloning movie

Cloning DNA: plasmid vectors

Origin of replication

Ampr gene (selectable)

Polylinker or multiple cloning site (MCS)

(Bacteriophages = alternative cloning vector)

Multiple cloning site

Region of plasmid containing multiple restriction enzyme sites to enable insertion of DNA of interest

Cutting DNA: restriction enzymes

Site specific endonucleases produced by bacteria

Recognize palindromic sequences (same 5’ --> 3’ on both strands)

Evolved to cleave bacteriophage DNA

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?- bacteria DNA is protected from cleavage by methylation

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

1. DNA (genomic, plasmid, PCR, ….)

2. DNA fragmentation/digestion

3. DNA Separation and purification

4. Forming recombinant DNA: ligation

5. Cloning DNA: Transformation,

selection and amplification

Essential steps in the generation of recombinant DNA

Forming recombinant DNA molecules: ligation

- T4 DNA ligase

-Requires ATP

-Phosphodiester bond

-Ligation of sticky ends is more efficient than blunt

Cloning DNA molecules: transformation, selection and amplification

1. Transformation = Introduction of plasmid into bacteria- Make “competent” bacteria- Add DNA- Inefficient uptake

2. Selection for antibiotic resistance

3. Amplification: Bacteria replicate w/ plasmid

Other Methods in recombinant DNA technology

Southern blot

DNA sequencing

Southern Blot

Microarray technology evolved from Southern blotting

DNA Sequencing: dye terminator sequencing

This week’s lab:

PCR

Restriction Digests

Agarose Gel Electrophoresis