Recombinant DNA

Enzymes:

• enzymes are protein-based molecules that facilitate various biochemical reactions, including the cutting and joining of DNA strands

• enzymes have an active site, which is where they bind with other molecules to combine or decompose them.

• what are restriction enzymes?

  • enzymes that cut double-stranded DNA at a unique, specific place

• two types of restriction enzymes:

  • endonuclease - makes cuts within the DNA molecule

    • three types - type II is used in rDNA technology as it makes cuts within recognition sites

      • examples: Eco R1, Hind III

  • exonuclease - removes nucleotides from the ends of the DNA strand

• why the name restriction enzyme?

  • the enzymes of bacterium restrict the replication of viral genome

• how do restriction enzymes cut DNA molecules?

  • recognition sites - DNA sequence of base pairs that reads the same on two strands in the 5’-3’ & 3’-5’

What is Recombinant DNA

• DNA fragments that are extracted from an organism and then inserted into another organism to change the genome and phenotype.

How Does Recombinant DNA Work?

• isolation - DNA is extracted from the source organism, and specific genes are targeted.

• cutting DNA - restriction enzymes cut the DNA at specific sequences, creating fragments with blunt and sticky ends.

• insertion - the DNA fragment is inserted into a vector, such as a plasmid or virus, which serves as a carrier for the DNA.

• ligation - DNA ligase (‘lig-’ means ‘tie’) seals the DNA fragments into the vector, creating recombinant DNA.

• transformation - the recombinant DNA is introduced into a host organism, such as a bacterium, using heat shock or electroporation techniques.

• replication and expression - the recombinant DNA replicates and expresses the desired trait inside the host.

What is the Point of Recombinant DNA?

• to manipulate what millions of years of evolution have allowed us to do

  • allows us to change genomes for all reasons

    • improve our immune systems to make us healthier

    • give organisms desired traits

• medicine

  • production of insulin and vaccines efficiently and at a low cost

• agriculture

  • development of genetically modified organisms

    • improved yield, pest resistance, and/or nutritional value.

• research

  • replication of organisms to study viruses, diseases, and genetics

• industry

  • manufacturing enzymes for detergents and biofuels.

Bacterial Transformations

Why Transform Bactiera?

• genetic engineering

  • the old - the domestication of plants and animals through farming and selective breeding

  • the new - the direct alteration of an organism’s genetics to achieve desirable traits through the process of genetic engineering

• genetic engineering involves the use of recombinant DNA technology

  • the recombinant DNA is then introduced into a host organism

The Tools of rDNA

• plasmids - vectors used to transfer DNA

  • double-stranded DNA that replicates independently of the bacterial chromosome

  • can be genetically engineered

• selectable marker

  • help researchers separate bacteria carrying the plasmid from bacteria not carrying the plasmid

  • most commonly antibiotic-resistant

• recognition sites

• ori region