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Basic DNA Science
BE Bootcamp 2008
Phillips Group / Caltech
Three Molecules of Life
DNA: four nucleotide bases (GC,AT) (2 bits)
genetic code in 3 base ‘codons’
information storage and propagation,
genetic regulation
Protein: folded polypeptide of 20 amino acids
motility, metabolism, reproduction,
genetic regulation, transport, etc.
Lipid: polar / non-polar molecules
separate ‘self’ from ‘non-self’
regulate material flow, cell shape,
compartmentalizes, etc
Three Molecules of Life
DNA: four nucleotide bases (GC,AT) (2 bits)
genetic code in 3 base ‘codons’
information storage and propagation,
genetic regulation
Protein: folded polypeptide of 20 amino acids
motility, metabolism, reproduction,
genetic regulation, transport, etc.
Manipulating DNA – Protein Relationships:
Revolutionized biological research (e.g. crystallography,
fluorescent proteins as markers) and medicine (e.g. drug
manufacture)
The Central Dogma
1) A simplified model.
2) Played out across life.
3) Many distinct points
for control.
Say Hello to Our Little Friend
Escherichia coli
Genome: circular, long (5 Mbp / 1.25 Mb)
difficult to manipulate
Plasmid: circular, short (1 - 5 kbp / 1.25 kb)
easy to manipulate
Say Hello to Our Little Friend
Escherichia coli
1)
2)
3)
4)
How do we get the gene of interest onto the plasmid?
How do we get the plasmid into the bacterium?
How do we convince the bacterium to use this DNA?
How do we tell if genes are transcribed?
The Alpha and the Omega
Genotype
Phenotype
E. coli expressing protein
-galactosidase
E. coli expressing fluorescent
protein from jellyfish (YFP)
Awesome, but Imperfect Tools
Cloning
Vector
plasmid purification / (double) restriction digest
Insert
/
PCR
gel purification
Awesome, but Imperfect Tools
Cloning
Vector
plasmid purification / (double) restriction digest
/
gel purification
Vector +
Insert
Insert
PCR
Ligation
Transform (Electroporation)
Plasmid Structure
Direction of transcription
Direction of transcription
pZE21-Venus(YFP)
Promoter – RNA polymerase binding site,
transcriptional regulator
Origin of Replication – site where plasmid
replication begins for division, controls
copy number and hence regulates
Restriction Sites – sequence-specific
enzymatic DNA cleavage sites, leaves
sticky ends for proper insert ligation
Kanamycin – encodes gene for
Kanamycin (fungal) antibiotic resistance,
imparts severe selective advantage in
proper media
Non-descript DNA – contain other
restriction sites for gene insertion
Plasmid Structure
pZE21-LacZ
pZE21-Venus(YFP)
HindIII
KpnI
Polymerase Chain Reaction
High temp (98C)
DNA denatures
Forward Primer
Reverse Primer
Lower temp (62C)
Primers anneal
Polymerase Chain Reaction
Lower temp (62C)
Primers anneal
Free nucleotides
Raise temp (72C)
Polymerase extends DNA
Polymerase Chain Reaction
35 cycles = 1011
Plasmid Restriction
HindIII
KpnI
Vector / Insert Ligation
Vector + Insert + Ligase
fluorescent cells
blue cells
white cells
Polymerase Chain Reaction