Recombinant DNA and biotechnology

Recombinant DNA and its applications

Gene cloning and plasmids
A plasmid is often used for gene cloning due to three key features:

1. A restriction site that can be cut open by a restriction enzyme, allowing insertion of the gene of interest.
2. An origin of replication, enabling the plasmid (and the cloned gene within) to replicate independently of the host’s genome.
3. An antibiotic resistance gene, allowing only bacteria containing the plasmid to survive under antibiotic treatment.

During cloning, restriction enzymes (endonucleases) cut double-stranded DNA at palindromic sequences, producing either sticky ends (which can reanneal) or blunt ends (which cannot). The resulting construct is introduced into host cells, which replicate the plasmid, thereby cloning the inserted gene.

DNA libraries
Researchers generate DNA libraries by cloning an organism’s entire DNA set:

• Genomic library: Created by fragmenting all genomic DNA with restriction enzymes and cloning the pieces into vectors.
• cDNA library: Made by reverse-transcribing the mRNA population into complementary DNA (cDNA), then cloning those cDNA fragments. This library reflects only expressed genes.

Hybridization

• Hybridization (annealing) refers to single-stranded DNA molecules binding to complementary sequences. For instance, a DNA probe used in Southern blot analysis anneals to a matching target DNA fragment on a gel. In gene cloning, sticky ends on a gene fragment can hybridize with sticky ends of a plasmid.

PCR (polymerase chain reaction)
PCR amplifies DNA exponentially via repeated cycles:

1. Denaturation: Heating to separate strands.
2. Annealing: Cooling so primers bind to their complementary sequences.
3. Elongation: A heat-stable polymerase extends the primers in the 5′→3′ direction.
   Doubling occurs each cycle ($2^n$).

Gel electrophoresis and blotting

• Gel electrophoresis separates DNA fragments by size within a gel matrix.
• Southern blot uses a labeled DNA probe to detect a specific fragment on the gel.
• Other blots include Northern (RNA target, DNA or RNA probe) and Western (protein target, antibody probe).

DNA sequencing

1. Sanger sequencing uses dideoxy-NTPs for chain termination and can be detected by capillary electrophoresis. It’s economical for short sequences but limited in scope.
2. Next-generation sequencing conducts massive parallel reactions, ideal for entire genomes or detecting low-level variants, but is more expensive.

Gene expression analysis

• mRNA levels reflect gene expression.
• Microarray (gene expression profiling) reveals which genes are overexpressed or underexpressed globally (e.g., in cancer).
• qRT-PCR (quantitative reverse transcription PCR) measures expression of a specific gene, such as residual BCR-ABL transcripts in leukemia.

Stem cells

• Stem cells can differentiate into many cell types. For example, hematopoietic stem cells can yield various blood cells. Clinically, a bone marrow transplant harnesses these stem cells to treat leukemia.

Practical applications of DNA technology

• Medical: PCR plus sequencing can diagnose mutations, predict drug responses, or monitor diseases.
• Gene therapy uses viruses to insert functional genes into human cells.
• Pharmaceuticals: Recombinant proteins or enzymes to treat deficiencies.
• Forensics: DNA fingerprinting for paternity tests or criminal identification.
• Environmental: Genetically engineered bacteria to degrade oil spills or plastics.
• Agriculture: GMO crops with improved yields or pest resistance.

Safety and ethics

• Gene therapy can inadvertently introduce mutations causing cancer.
• Genetically modified foods have potential to limit the diversity of edible plant species and create susceptibility in the food supply, and their possible negative long term effects on human health have not been absolutely ruled out.
• Full genome sequencing raises privacy and discrimination concerns regarding genetic data.

Determining gene function

Understanding how a particular gene contributes to biological processes often involves a combination of comparative genomics, protein domain analysis, interaction studies, and expression profiling. These methods, taken together, help to illuminate a gene’s role within the cell or organism.

1. Evolutionary comparison
   • Cross-species conservation: Researchers examine whether a gene is conserved among diverse organisms. Genes shared across multiple species, especially at the protein level, often serve fundamental roles. These sequence similarities can pinpoint regions critical for activity or stability.
   • Phylogenetic analysis: By reconstructing evolutionary relationships, scientists identify when a gene or its variants first appeared, providing clues about its earliest function and importance.
2. Protein domains
   • Domain architecture: Many proteins contain characteristic structural segments (e.g., kinase domains, DNA-binding motifs) that predict how they interact with other molecules. Recognizing such domains helps infer whether a protein might be involved in signaling, catalysis, or nucleic acid binding.
   • Functional predictions: If a newly discovered protein shares a domain with a well-studied protein family, it may carry out comparable tasks—even if the organism is different.
3. Protein interactions
   • Interaction networks: Determining which partners a protein binds or associates with can reveal its functional context. Techniques such as co-immunoprecipitation, yeast two-hybrid, or mass spectrometry-based proteomics identify interaction partners that co-regulate pathways or physically assemble into protein complexes.
   • Pathway assignment: If a protein interacts with several known players of a specific metabolic or signaling cascade, it likely participates in that pathway.
4. Cellular expression
   • Spatial and temporal patterns: Monitoring the abundance and location of mRNA or protein under various conditions sheds light on where and when a gene is active. Techniques like in situ hybridization, immunofluorescence, and reporter assays reveal tissue-specific or developmental-stage-specific expression.
   • Perturbations and phenotypes: When expression is altered—through gene knockdowns, knockouts, or overexpression—changes in cellular or organismal traits can point directly to a gene’s normal role.