Cloning and Expression of Genes

• Insulin: Harmone regulating blood sugar (glucose)

• Obtain Insulin Gene from Human DNA• Insert gene in bacterial cells• Select cells that have desired gene• Induce bacteria to express foreign gene to

produce insulin• Collect and purify insulin

Isolating the Gene

• Isolate mRNA• Copy to DNA with reverse transcriptase• cDNA (copy or complementary DNA)

Inserting the Gene

• Carrier DNA is a vector• Plasmids: Circular DNA found in

microorganisms• Cloning Vector• Easily transferred, incorporated, and isolated• Inserted with restriction enzymes• Cut up foreign DNA

Selecting Cells with Desired Gene

• Most cells don’t take up DNA• Ampicillin resistance gene in plasmid

vectors• Only cells with plasmids grow• Blue-White Screening for gene or not• Intact bacteria gene or not, Blue or White

Isolating the Insulin

Analysis of DNA

• Gel Electrophoresis• DNA Sequencing• Hybridization• Polymerase Chain Reaction• DNA Chips• Human Genome Project

DNA LIGATION

’ ’

’ ’

Ligase Joins 5' phosphateto 3' hydroxyl

’ ’

Gel Electrophoresis• Sort DNA by Size• Electrophoresis is separation of particles in an electric field

based on charge• Thin Agarose Gels: Matrix of interlocking fibers• DNA has negative charge• Repates through matrix• Ethidium Intercalator for visualization• Entanglement and speed related to charge and size• Restriction Map: Different restriction enzymes, different size

maps, ID restriction sites

GEL ELECTROPHORESIS - SIZE SORTING

BufferGel

Electrode

Electrode

Samples

Faster

Slower

RESTRICTION ENDONUCLEASES

EcoRI

HindIII

AluI

HaeIII

- OH 3’

5’ P -

- P 5’

3’ OH -

Sequencing

• Isolate fragment to be sequenced as SS• 4 Tubes, 4 Bases• Add mix of nucleotides, DNA polymerase, and

primers with radioactive labels• In each tube as single base is modified

(dideoxynucleotide)• Once added no other nucleotides added• Chain Termination Sequencing

DNA Sequencing

Hybridization

• Binding of complementary DNA strands to each other

• Find insulin sequence? Need prior information

• Hybridize with part of the insulin sequence

Gene Expression

• Study production of mRNA in the cell• Clone genes of interest• Make cDNA probes • Sample mRNA populations of cell• Detect by hybridization to radioactive probe

Polymerase Chain Reaction

• Amplify a single piece of DNA • Makes a large number of copies• Target specific primers allow specific

sequences to be amplified

DNA Polymerase

POLYMERASE CHAIN

REACTION

DNA Arrays

• DNA Sequences Affixed to Solid Support• Genotyping and Gene Expression• Entire Genomes on Chips• Look at Relative Intensity Levels• Massively Parallel• Loads of Data• Interpretation, Storage, Analysis?

http://www.affymetrix.com

http://cmgm.stanford.edu/pbrown/

ANTIBODY AFFINITY

CACCATGTGAC

GTGGTACACTG B

PMP

+

Anneal

CACCATGTGAC

GTGGTACACTG B+

CACCATGTGAC

GTGGTACACTG B PMP

Bind

Add oligo withBiotin label

Heat and cool

Add Paramagnetic-Streptavidin

Particles

Isolate with MagnetN

S

Mapping and Sequencing the Human Genome

• Find all genes in Human Genome• Develop Tools for Analysis in Biology and

Medicine• Tools:

– Restriction Enzymes– PCR– DNA Arrays