Methods of Obtaining Genes
To create a recombinant molecule, the gene of interest must first be obtained in sufficient quantities. This can be accomplished through two main pathways:
- Reverse transcription. Retroviral enzymes—reverse transcriptases—are used. They catalyze the synthesis of a DNA molecule using mRNA as a template.
- Chemical synthesis. Automated synthesizers are used to create single-stranded DNA with a predefined sequence. The main limitation of this method is that the fragment length must not exceed 100 nucleotides.
The Role of Restriction Endonucleases
Restriction endonucleases (restriction enzymes) are bacterial enzymes that act as molecular "scissors." Their primary function is to recognize short, specific nucleotide sequences (restriction sites) and cleave both DNA strands at these sites.
There are hundreds of restriction enzyme varieties, each specific to a particular site. Cleavage can produce two types of ends:
- Blunt ends — the enzyme cuts both strands directly opposite each other.
- Sticky ends — the cut occurs with an offset. This generates single-stranded overhangs capable of binding to other fragments via complementary base pairing.
Steps in Creating Recombinant DNA
The synthesis of a hybrid molecule relies on using restriction enzymes that generate "sticky ends" (e.g., Eco RI). The process includes the following steps:
- Preparation. The target gene and the vector DNA (e.g., a bacterial plasmid or viral DNA) are cut with the same restriction enzyme. This is necessary to generate identical complementary "sticky ends" in both molecules.
- Hybridization (Annealing). The single-stranded overhangs of different fragments pair with each other due to the complementarity of nitrogenous bases.
- Ligation. The enzyme DNA ligase covalently seals the fragments, fully restoring the sugar-phosphate backbone. The final product is the ready recombinant DNA.