Biotechnology and Recombinant DNA

What is Biotechnology

• Biotechnology– Use of microorganisms, cells or cell

components to make a product• Recombinant DNA technology (rDNA)

– Genetic engineering– Insertion of genes into cells that makes the

cells into “factories” to make products

Recombinant DNA

• Putting a gene from one organism into another

• Examples:– Human insulin gene into a bacteria to make

insulin– Hepatitis B gene into a yeast to make the

hepatitis B vaccine

How to make rDNA

• Gene of interest is inserted into a VECTOR

• Vector is usually a plasmid that must be self-replicating

• Cells containing the vector with the gene of interest then divide to from a CLONE of identical cells

• These clones can then be used to harvest the gene or produce a product

Restriction enzymes

• DNA cutting enzymes that are a key to the development of rDNA technology

• Discovered in the early 1970’s• Nobel Prize in 1978 to Arber, Nathans and

Smith for the discovery of these enzymes• Restriction enzymes cut DNA at specific

sites and allow for DNA to be “inserted” into a cloning vector

• “Sticky ends” are generated?

Restriction Enzymes

Vectors

• DNA molecules that can be used as transfer vehicles to insert DNA into cells

• Must be self-replicating and small enough to work with outside the cell

• Plasmids are common vectors• Often contain antibiotic resistance gene • Viral DNA is also used as a vector

– Retroviruses, Adenoviruses, Herpesviruses• Larger amounts of DNA can be inserted

Cloning plasmid

Kary Mullis

• Invented the technique of polymerase chain reaction in the early 1980

• Nobel Prize in 1993• Key technique used

to make large quantities of DNA

Polymerase chain reaction

Inserting DNA into cells

• 1. Transformation• 2. Electroporation

– Electric current make pores in the cell so DNA can enter

• 3. Protoplast fusion– Cells with no cell wall can be fused and

natural recombination may occur• 4. Microinjection

Protoplast fusion

How are genes isolated?

• 1. Gene libraries– Digestion of entire genome with restriction

enzymes– Insert fragments into vectors and put the

vectors into bacterial cells• 2. Complementary DNA (cDNA)

– Eukaryotic gene derived from mRNA made with reverse transcriptase

– Lacks introns only exons

Gene Library

Complementary DNA

Selecting the clone

• Need to next be able to find the cell with the gene of interest

• Selecting the clone of interest is often done with marker genes

• Genes are spliced into plasmids carrying genes for ampicillin resistance and β-galactosidase

• Colonies that grow with special characteristics are selected as potential clones with the gene you want!

Selecting recombinant bacteria

Selecting the clone

• After candidate colonies are identified the one with the gene of interest must be selected

• DNA probes• Pieces of single stranded DNA

complementary to the desired gene are made and labeled with a radioactive element

• Helps identify the target gene

DNA Probes

Making a gene product

• Put the gene into a bacteria like E. coli and get the product made– Toxic by-products from Gram- cell wall– Usually no secretion of product by Gram - cells

• Yeast cells– Better secretion of product

• Mammalian cells– Good source for protein products – Little risk of toxins and allergy

Products of genetic engineering

Products of genetic engineering