basic dna science be bootcamp 2008 phillips group / caltech
TRANSCRIPT
Basic DNA Science
BE Bootcamp 2008BE Bootcamp 2008Phillips Group / CaltechPhillips 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
Manipulating DNA – Protein Relationships:Revolutionized biological research (e.g. crystallography, fluorescent proteins as markers) and medicine (e.g. drug manufacture)
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.
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) How do we get the gene of interest onto the plasmid?2) How do we get the plasmid into the bacterium?3) How do we convince the bacterium to use this DNA?4) How do we tell if genes are transcribed?
The Alpha and the Omega
E. coli expressing protein -galactosidase
E. coli expressing fluorescent protein from jellyfish (YFP)
Genotype
Phenotype
Awesome, but Imperfect Tools
plasmid purification / (double) restriction digest / gel purification
Vector
Insert
Cloning
PCR
Awesome, but Imperfect Tools
plasmid purification / (double) restriction digest / gel purification
Vector
Insert
Cloning
PCR Ligation Transform (Electroporation)
Vector + Insert
Plasmid Structure
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
Direction of transcription
Direction of transcription
Plasmid Structure
pZE21-LacZ
pZE21-Venus(YFP)
KpnI
HindIII
Polymerase Chain Reaction
High temp (98C)DNA denatures
Forward PrimerReverse Primer
Lower temp (62C)Primers anneal
Polymerase Chain Reaction
Lower temp (62C)Primers anneal
Raise temp (72C)Polymerase extends DNA
Free nucleotides
Polymerase Chain Reaction
35 cycles = 1011
Plasmid Restriction
KpnI
HindIII
Vector / Insert Ligation
fluorescent cells blue cells white cells
Vector + Insert + Ligase
Polymerase Chain Reaction