Methods for obtaining genes
To create a recombinant molecule, one must first obtain the gene of interest in sufficient quantity. This can be achieved through two main pathways:
- Reverse transcription. Retroviral enzymes — reverse transcriptases — are used. They are capable of catalyzing 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.
Role of restriction enzymes
Restriction enzymes (endonucleases) are enzymes of bacterial origin that act as molecular "scissors." Their primary function is to recognize short specific nucleotide sequences (restriction sites) and cleave both DNA strands at these locations.
There are hundreds of varieties of restriction enzymes, each specific to a certain site. Cutting can result in two types of ends:
- "Blunt" ends — the enzyme cuts both strands directly opposite each other.
- "Sticky" ends — the cut occurs with an offset. As a result, single-stranded overhangs are formed that can bind to other fragments via the principle of complementarity.
Stages of recombinant DNA creation
The synthesis of a hybrid molecule is based on the use of restriction enzymes that form "sticky" ends (e.g., Eco RI). The process includes the following steps:
- Preparation. The target gene and vector DNA (such as a bacterial plasmid or viral DNA) are cut with the same restriction enzyme. This is necessary to form identical complementary "sticky" ends in both molecules.
- Hybridization (annealing). Single-stranded tails of different fragments pair with each other due to nitrogenous base complementarity.
- Ligation. The enzyme DNA ligase covalently joins the fragments, fully restoring the sugar-phosphate backbone. The output is ready recombinant DNA.
Briefly: answers to topical questions
Which molecules are used as vectors for creating recombinant DNA?
The following molecules are used as vectors for creating recombinant DNA:
- Plasmids — bacterial extrachromosomal circular DNA molecules.
- Bacteriophage DNA or viral DNA.
Vectors are necessary to introduce the foreign gene into the bacterium and ensure replication, transcription, and translation of the inserted gene.
In addition to reverse transcription and chemical synthesis, what other methods are used to obtain target genes?
In addition to reverse transcription and chemical synthesis, methods for isolating (cloning) natural genes are used to obtain target genes:
- Isolation of entire genetic structures (chromosomes, plasmids).
- Enzymatic cleavage — the use of restriction enzymes that cut DNA at strictly defined bonds.
- RNA cleavage — the use of ribozymes that act on RNA similarly to restriction enzymes.
To remember the types of ends in restriction: A "blunt" end cuts straight (without overhangs), while a "sticky" end cuts with a shift, leaving a tail to "stick" to its neighbor.
Class and exam questions
Which enzymes synthesize DNA using an mRNA template?
This reaction is catalyzed by reverse transcriptases (retroviral enzymes).
How do "blunt" ends differ from "sticky" ends?
During the formation of "blunt" ends, DNA strands are cut directly opposite each other, whereas during the formation of "sticky" ends, they are cut with an offset, yielding single-stranded complementary tails.
Which enzyme joins DNA fragments?
Covalent joining of fragments and restoration of the sugar-phosphate backbone is performed by DNA ligase.
What else to study on this topic
- Mechanism of action of the Eco RI enzyme
- The process of molecular cloning in bacterial cells
- Specificity of restriction sites
- Vector systems: plasmids, bacteriophages, viruses
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