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