Recombinant DNA Technology: Methods and Applications
Restriction Enzymes
Saccharomyces cerevisiae plays a crucial role in recombinant DNA technology. This process involves coding sequences and regulatory elements. Restriction endonucleases, bacterial enzymes capable of cutting DNA, are fundamental. They recognize specific palindromic sequences, producing either blunt or cohesive ends. DNA fragments are then separated by gel electrophoresis, using agarose or polyacrylamide gels.
Plasmids and DNA Ligases
Plasmids, bacterial or viral genomes, and artificially synthesized DNA molecules serve as vectors. DNA ligases create covalent bonds between DNA fragments. Complementary DNA (cDNA), synthesized from an RNA template, lacks introns, regulatory regions, or repetitive sequences found in genomic DNA.
De Novo DNA Synthesis
DNA oligonucleotides are synthesized using a 3′ silica gel support. The oligonucleotides are finally separated by centrifugation or filtration.
Hybridization
Hybridization is based on identifying genes through the mating ability of single-stranded nucleic acid probes. Two main types exist: DNA-DNA (Southern blot) and DNA-RNA (Northern blot).
Genomic Libraries
Genomic libraries are collections of DNA fragments introduced into bacteria for storage, saving time. Complementary DNA is often used for this purpose.
Polymerase Chain Reaction (PCR)
PCR allows for the amplification of specific DNA fragments. It requires DNA polymerase, a suitable primer, a DNA template, and deoxynucleotide triphosphates. The process involves denaturation at high temperatures and is exponential. Thermophilic bacteria are often the source of heat-stable DNA polymerases. PCR has applications in archaeology, criminology, paleontology, and medicine.
Cloning Process
The cloning process involves these steps:
- Isolation and preparation of the gene to be cloned.
- Selection of an appropriate cloning vector.
- Formation of recombinant DNA.
- Introduction of recombinant DNA into a host cell.
- Verification of cloned gene expression.
Cloning Vectors
Bacterial Cells
Restriction endonucleases are used to create compatible ends for ligation. Plasmids, commonly found in bacteria, are smaller than chromosomes and have their own origin of replication. Viral genomes, such as those of phage alpha and M13, also serve as vectors. Artificial chromosomes with markers, like antibiotic resistance genes, are also employed.
Eukaryotic Cells
Cloning in eukaryotic cells is more complex due to the presence of introns and the regulated nature of gene expression. Transgenic organisms are created by inserting genes into embryonic cells. Gene therapy involves modifying genes in somatic cells.
Cloning Vectors for Eukaryotes
Viral Genomes
Some viruses integrate their DNA into host chromosomes (e.g., retroviruses). Virulence genes are often eliminated. Vaccinia virus and adenovirus are used for temporary gene expression.
Artificial Chromosomes
Yeast artificial chromosomes (YACs) are unique to yeast and are maintained stably in the nucleus. They contain a yeast origin of replication, a centromere, and two telomeres, allowing them to carry very large DNA fragments.
Plasmid Ti
Agrobacterium tumefaciens, a bacterium that causes tumors in plants, has a Ti plasmid used for gene transfer. The T-DNA and virulence genes are often deleted for cloning purposes.
Selection of Host Cells
Several methods are used to introduce the vector into the host cell:
- Microinjection: A very small diameter capillary is used to inject DNA directly into the cell membrane, often used in embryos.
- Electroporation: High-voltage electrical impulses permeabilize the cell membrane, facilitating DNA entry.
- Gene Gun: DNA-coated microprojectiles are propelled into cells.
- Liposomes: Lipid bilayer vesicles containing DNA fuse with the cell membrane.
- Agrobacterium tumefaciens Ti plasmids: Used for plant cell transformation.
Goals of Genetic Engineering
Genetic engineering aims to:
- Introduce new genes into a genome.
- Delete specific genes.
- Modify existing genetic information.
- Change patterns of gene expression.
- Clone living beings and organs.