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 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 PCRLigation 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

Plasmid Structure pZE21-LacZ pZE21-Venus(YFP) KpnI HindIII

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 Raise temp (72C) Polymerase extends DNA Free nucleotides

Polymerase Chain Reaction 35 cycles = 10 11

Plasmid Restriction KpnI HindIII

Vector / Insert Ligation fluorescent cells blue cellswhite cells Vector + Insert + Ligase

Polymerase Chain Reaction