18- Recombinant DNA Technology

Overview of Recombinant DNA Technology

• Recombinant DNA = combining DNA from different species → genes can cross species barriers.

Recombinant DNA Technology Use

• Used to isolate, recombine, transfer, and express DNA.

Recombinant DNA Technology Requires

• Tools: enzymes, vectors, host cells

• Techniques: isolation, characterization, manipulation

• Technology has major benefits & serious bioethical/safety considerations.

Restriction Endonucleases

• Key enzyme Isolated from prokaryotes; protect bacteria from viruses by cutting foreign DNA.

• Recognize specific DNA sequences and cleave at or near those sites.

• Types:

  • Type I & III: recognize one site but cut at another; also have methylase activity.

  • Type II: most used; cut at specific sequences; no methylase activity.

• Example: EcoRI recognizes 5’-GAATTC-3’.

• Cuts create:

  • Sticky ends (5’ or 3’ overhangs)

  • Blunt ends

• Essential for building recombinant DNA.

DNA Polymerase

• Key enzyme Used for in vitro DNA synthesis.

• Requires template, primers , dNTPs, Mg²⁺, proper buffer.

• Polymerases come from:

  • E. coli

  • T7 phage

  • Thermostable bacteria (PCR)

• Used for sequencing, PCR, DNA replication in vitro.

DNA Ligase

• Key enzyme, Covalently joins the 3’ and 5’ ends of DNA fragments.

• Works on both blunt and sticky ends.

• Example: T4 DNA ligase (from T4 phage).

Reverse Transcriptase (RT)

• Key enzyme

• RNA-dependent DNA polymerase → makes cDNA from RNA.

• Used for:

  • cDNA library construction

  • Amplifying DNA from RNA templates

• Examples: AMV RT, MMLV RT.

Plasmid Vectors

• Circular dsDNA with:

  • Origin of replication (ori)

  • Antibiotic resistance gene

  • Unique restriction sites

• Used for cloning smaller DNA

• Less efficient than phage vectors but easier and more stable.

• Examples: pBR322, pBluescript.

Phage Vectors

• Derived from lambda phage

• Insert DNA into non-essential region of phage genome.

• Phage infects E. coli (transfection) — more efficient than transformation.

Hosts

• E. coli → routine cloning

• Yeast → eukaryotic gene expression studies

• Plant cells & animal cells in culture for expression studies

Gel Electrophoresis

• Separates DNA/RNA by size using electric charge.

• DNA/RNA move toward positive electrode.

• Larger fragments = slower; smaller = faster.

• Agarose = for DNA/RNA (approximate separation).

• Polyacrylamide = high-resolution, sequencing gels.

RFLP (Restriction Fragment Length Polymorphism)

• Compares restriction patterns between individuals.

• DNA → cut → run on gel → compare fragment sizes.

• Differences indicate mutations in restriction sites.

Radiolabeling DNA

• Incorporate radioactive or fluorescent labels into DNA.

• Used in:

  • Sequencing

  • Southern/Northern blots

  • Library screening

DNA Sequencing (Sanger method)

• Uses:

  • Template DNA

  • Primer

  • DNA polymerase

  • dNTPs + fluorescently-labeled ddNTPs

• ddNTPs terminate synthesis → produce various fragment lengths.

• Fragments separated by polyacrylamide gel or capillary electrophoresis.

• Next-gen uses capillaries or nanopores.

PCR (Polymerase Chain Reaction)

• Amplifies DNA from tiny amounts.

• Uses two primers + thermostable polymerase.

• Steps:

  • Denaturation hot

  • Annealing

  • Extension

CRISPR/Cas

• Natural bacterial defense system recognizing viral DNA.

• Cas proteins cut unwanted DNA.

• Used to remove defective genes or viral remnants.

• Large ethical considerations, esp. human genome editing.

Genomic vs cDNA libraries

• Genomic library: includes introns, exons, promoters, noncoding DNA.

• cDNA library: contains only coding region (from processed mRNA using RT).

Library Screening

• Probe (radioactive or synthetic DNA) hybridizes to complementary DNA.

• DNA is:

  • Denatured → ssDNA

  • Neutralized → allowing annealing

• Hybridizing colonies identified through probe signal.

• Isolated clones → sequenced and characterized.

Genomics & Bioinformatics

• Structural genomics: DNA/protein sequence analysis.

• Functional genomics: gene function and expression.

• Bioinformatics = computational analysis of sequence data.

• Whole-genome sequencing requires genetic mapping first.

Southern Blot

• DNA-DNA hybridization.

• Determines:

  • Gene copy number

  • Genome organization

  • RFLP differences

  • Presence/absence of a gene

Northern Blot

• RNA-DNA hybridization.

• Measures mRNA levels in tissues, stages, or conditions.

Western Blot

• Protein-antibody recognition.

• Primary antibody → antigen

• Secondary antibody (fluorescent/enzyme-tagged) → detection.

Microarray Analysis

• Thousands of DNA samples on a slide.

• Fluorescent-labeled mRNA hybridizes → shows which genes are expressed.

Bacterial Transformation

• CaCl₂ treatment

• Electroporation (electric pulse; more efficient)

• Transfection with recombinant phages (library construction)

Plant Transformation

• Uses totipotent cells.

• Main method: Agrobacterium tumefaciens

  • Transfers recombinant plasmid → integrates into chromosome

• Hard-to-transform plants:

  • Gene gun / particle bombardment

• Selection via antibiotic resistance.

Animal Transformation

• Salt-mediated DNA uptake

• Electroporation

• Transfection

• Microinjection into large cells (e.g., frog oocytes)

• Whole-animal transformation:

  • Introduce DNA into embryos, eggs, or sperm → implant into surrogate.

Applications of Recombinant DNA Technology- Plants

• Benefits: herbicide resistance, insect resistance, disease resistance, improved storage.

• FDA-approved examples:

  • Flavr-Savr tomato (long shelf life)

  • Golden rice (beta-carotene enriched)

  • Insect-resistant crops

  • Herbicide-resistant crops

Applications of Recombinant DNA Technology- Animals

Used in:

• Diagnostics & vaccines

• Growth hormones (BST for milk/meat production)

• Early embryonic gene insertion for protein production

Applications of Recombinant DNA Technology- Humans

• Major uses:

  • Genetic disease identification

  • HIV diagnostics

  • Recombinant vaccines (multi-antigen plasmids)

  • Gene therapy

  • Forensics & paternity tests

• Recombinant insulin = first major biopharmaceutical success.

• mRNA COVID-19 vaccines = major modern achievement.

• Human Genome Project → identifies disease genes, enables targeted medicine.

Biological Safety

• NIH sets laboratory safety guidelines.

• Risks:

  • Pathogens

  • DNA mutagenic chemicals

  • Accidental release or contamination

• FDA regulates recombinant product safety.

• EPA tests environmental release of GMOs.

• USDA monitors GM crop testing/release.

Ethical Issues

• Major debates include:

  • Fetal tissue use

  • Human cloning (e.g., Dolly, Gene the cow raise concerns)

  • Genetic discrimination by employers/insurance companies

  • Use of embryonic vs adult stem cells

  • Manufacturing human organs in labs

• Laws exist to prevent misuse of genetic information.