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