Biotechnology: Principles, Processes and Applications
Biotechnology: Principles, Processes and Applications examines the tools of recombinant DNA technology and their products.
Core rules
- Restriction endonucleases recognise palindromic sequences and cut DNA. EcoRI cuts G^AATTC and leaves sticky ends. DNA ligase joins the sugar–phosphate backbone.
- A vector needs an origin of replication, selectable markers, and recognition sites. pBR322 carries ampicillin and tetracycline resistance; the Ti plasmid of Agrobacterium tumefaciens is used for plants.
- Insertional inactivation: an insert in the tetracycline gene disables it, so recombinants are ampicillin-resistant but tetracycline-sensitive.
- PCR: denaturation (~94 °C), annealing (~50–60 °C), extension (~72 °C) with Taq polymerase. Gel electrophoresis separates fragments by size.
- Applications: gene therapy (ADA deficiency), RNAi, Bt cotton, insulin, GEAC regulation, biopiracy concerns.
Worked example
Starting with one DNA molecule, how many copies after 15 PCR cycles? Each cycle doubles the number, so copies = 2^15 = 32,768. With 10 starting molecules the count is 10 × 32,768 = 327,680.
Traps
Taq polymerase is thermostable and is used in PCR, not in the human cell. Ligase forms phosphodiester bonds, not hydrogen bonds. Sticky ends are complementary, not identical. Selection on ampicillin alone cannot distinguish recombinant from non-recombinant plasmids.