explain the roles of restriction endonucleases, DNA ligase, plasmids, DNA polymerase and reverse transcriptase in the transfer of a gene into an organism

Principles of Genetic Technology

Learning Objective

Explain the roles of restriction endonucleases, DNA ligase, plasmids, DNA polymerase and reverse transcriptase in the transfer of a gene into an organism.

Key Molecular Tools in Gene Transfer

1. Restriction Endonucleases

Restriction endonucleases are enzymes that recognise specific short DNA sequences (usually 4–8 bp) and cleave the phosphodiester backbone within or near these sites. They generate:

• Sticky ends – over‑hanging single‑stranded DNA that can base‑pair with complementary sequences.
• Blunt ends – straight cuts with no over‑hangs.

In recombinant DNA technology, they are used to cut both the vector (e.g., a plasmid) and the gene of interest, producing compatible ends that facilitate ligation.

2. DNA Ligase

DNA ligase catalyses the formation of phosphodiester bonds between adjacent nucleotides. After restriction enzymes generate compatible ends, DNA ligase joins:

• The 5′‑phosphate of one DNA fragment to the 3′‑hydroxyl of another.
• Both sticky‑ended and blunt‑ended fragments, although sticky ends ligate more efficiently.

This step creates a stable recombinant DNA molecule.

3. Plasmids

Plasmids are circular, double‑stranded DNA molecules that replicate autonomously in bacterial cells. Important features for gene transfer include:

• Origin of replication (ori): ensures plasmid duplication.
• Selectable marker genes: e.g., antibiotic resistance, allowing identification of transformed cells.
• Multiple cloning site (MCS): a region containing several restriction sites for easy insertion of foreign DNA.

Plasmids serve as vectors that carry the gene of interest into a host organism.

4. DNA Polymerase

DNA polymerases synthesise new DNA strands complementary to a template. In gene transfer they are used in two main contexts:

• Polymerase Chain Reaction (PCR): amplifies the target gene to obtain sufficient quantity for cloning.
• Repair of nicks: after ligation, DNA polymerase can fill in missing nucleotides at sticky‑end junctions before ligase seals the backbone.

5. Reverse Transcriptase

Reverse transcriptase synthesises complementary DNA (cDNA) from an RNA template. Its roles in gene transfer include:

• Conversion of eukaryotic mRNA (which may contain introns) into intron‑free cDNA suitable for expression in prokaryotic hosts.
• Generation of cDNA libraries for screening and cloning of specific genes.

Step‑by‑Step Overview of Gene Transfer

1. Isolate the gene of interest (often by PCR).
2. Use restriction endonucleases to cut both the gene fragment and the plasmid vector at compatible sites.
3. Purify the fragments and mix them to allow sticky ends to anneal.
4. Add DNA ligase to seal the phosphodiester bonds, forming recombinant plasmid DNA.
5. Introduce the recombinant plasmid into a host cell (e.g., bacterial transformation).
6. Select transformed cells using the plasmid’s selectable marker.
7. Confirm insertion by colony PCR, restriction analysis, or sequencing.

Summary Table

Key Equations (LaTeX)

During PCR amplification, the amount of DNA after n cycles is given by:

\$N = N_0 \times 2^{n}\$

where \$N_0\$ is the initial copy number and \$N\$ is the final copy number.

For ligation efficiency, the probability \$P\$ of successful ligation of two sticky‑ended fragments can be approximated by:

\$P = 1 - e^{-k [\text{DNA}] t}\$

where \$k\$ is the rate constant, \$[\text{DNA}]\$ the concentration of DNA ends, and \$t\$ the incubation time.