Genetic Research and Biotechnology Recombinant technology

Selection of Traits Artificial selection - breeding; novel crosses Recombinant DNA technology Selection of Traits Artificial selection - breeding; novel crosses Recombinant DNA technology - DNA from one organism cut and spliced (recombined) with DNA of another Genetic engineering - gene isolation, modification and insertion into original or different organism

Recombinant DNA technology Recombinant DNA - DNA which has been altered by joining genetic material from two different sources. It usually involves putting a gene from one organism into the genome of a different organism, generally of a different species. ‘Typical ‘ bacteriophages have a hexagonal head, tail and tail fibers Attach to cell wall of a host cell with tail fibers Alter the cell wall of host cell inject nuclear material into host cell Werner Arber proposed that restricted growth of phages occurred because some bacteria contained enzymes that could cut viral DNA into small pieces, thus preventing viral replication. http://www.pbs.org/faithandreason/biogloss/recomb-body.html

Bacterial and Viral Battles Some bacteria and viruses insert their DNA into other cells that are to act as hosts Bacteria use restriction enzymes to cut up foreign DNA These characteristics are useful in recombinant technology

Roots of Recombinant Technology Plasmids are small, circular pieces of DNA in bacteria, that replicate independently of the bacterial chromosome Pieces of foreign DNA can be added to plasmids to create recombinant DNA Replication can produce 50 – 100 copies of the recombinant plasmid

Recombinant DNA technology Restriction enzymes cut DNA by cleaving the phosphodiester bond in the DNA strand.

Like other enzymes restriction enzymes show specificity for certain substrates, and will only digest DNA within specific sequences of bases - called recognition sequence or a restriction site. Some restriction enzymes cut DNA into overhanging single stranded ends. Others will generate fragments with double-stranded non-overhanging ends called ‘blunt ends’.

Recombinant DNA technology Recombinant DNA refers to DNA which has been altered by joining genetic material from two different sources. It usually involves putting a gene from one organism into the genome of a different organism, generally of a different species. DNA from any source such as bacteria, humans, dogs, cats, frogs, dinosaurs, can be digested. How would you put cut DNA back together?? DNA ligase Paul Berg founded recombinant DNA technology when he spliced together DNA from E.coli chromosome and DNA from a primate virus.

Recombinant DNA technology Restriction enzymes cut double stranded DNA at specific sites These cuts produce DNA fragments that can be fit back together Ligase is like glue, and rejoins the DNA.

Cohen also made another important contribution to gene cloning – he demonstrated transformation: a process for inserting foreign DNA into bacteria. use of calcium chloride solution and temperature change, or electroporation compromise the bacterial membrane enough to allow plasmid DNA to enter the cells. Antibiotic selection can be used to select the transformed gene.

So what?? How would you use this?? Summary We can : cut DNA (what do we use??) paste it back together (what do we use??) put it into a vector (what does this mean??) get the vector into a cell of interest(why)?? So what?? How would you use this??

Cloning Requires a Vector… Gene Cloning in Bacteria Cloning …specialized DNA technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough material for further study. Requires a Vector… ...a specialized DNA sequence that can enter a living cell, ...signal its presence to an investigator, ... and provide a means of replication for itself and the foreign DNA it carries.

And, Vectors… …contain unique restriction sites to facilitate the creation of recombinant DNA molecules, ... must also possess a distinguishing physical characteristic such as size or shape by which it can be purified away from the host cells genome.

Step 1…restriction digests and ligation of fragments into cloning vectors,

Cloning Step 2 …vector-insert recombinants are inserted in host cells, Transformation (via bacterial mechanisms) Transduction (via viral mechanisms)

Cloning Step 3 ...vectors contain selectable markers, only cells that contain vector DNA will survive selection, recombinant vectors can be discerned from empty vectors by additional markers. Blue/White Cloning

Amplification …if you were to grind me up with a giant mortar and pestle, and extract all of my DNA coding for a single gene, you’d get about 1 mg of DNA, …two liters of bacterial culture carrying my gene in a recombinant DNA vector, would yield an equivalent mass of DNA.

Bacterial Production of Human Insulin The final steps are to collect the bacteria, break open the cells, and purify the insulin protein expressed from the recombinant human insulin gene