explain that genes to be transferred into an organism may be: extracted from the DNA of a donor organism, synthesised from the mRNA of a donor organism, synthesised chemically from nucleotides

Published by Patrick Mutisya · 14 days ago

Cambridge A-Level Biology – Principles of Genetic Technology

Principles of Genetic Technology

Objective

Explain that genes to be transferred into an organism may be:

  • Extracted from the DNA of a donor organism

  • Synthesised from the mRNA of a donor organism

  • Synthesised chemically from nucleotides

1. Extraction of Genes from Donor DNA

DNA is isolated from the cells of the donor organism and the gene of interest is cut out using restriction enzymes. The steps are:

  1. Cell lysis to release genomic DNA.

  2. Use of specific restriction endonucleases to cleave at known sequences flanking the target gene.

  3. Separation of the fragment by agarose gel electrophoresis.

  4. Purification of the DNA fragment for insertion into a vector.

2. Synthesis of Genes from Donor mRNA (cDNA)

When the gene is expressed in the donor, its mRNA can be reverse‑transcribed to produce complementary DNA (cDNA). This method is useful for genes that contain introns, because the mRNA is already spliced.

  1. Isolate total RNA from the donor tissue.

  2. Use reverse transcriptase to synthesise cDNA from the mRNA template.

  3. Amplify the cDNA by polymerase chain reaction (PCR) using gene‑specific primers.

  4. Clone the amplified cDNA into a suitable vector.

3. Chemical Synthesis of Genes

Advances in solid‑phase DNA synthesis allow a gene to be built nucleotide by nucleotide from basic chemical building blocks. This approach is valuable when:

  • The exact sequence is known and can be optimised (e.g., codon optimisation for a new host).

  • The gene is not readily available from a natural source.

  • Specific mutations or tags need to be introduced during synthesis.

The general steps are:

  1. Design the desired nucleotide sequence.

  2. Automated synthesis of short oligonucleotides (oligos).

  3. Assembly of oligos into the full‑length gene using ligation or PCR‑based methods.

  4. Verification of the assembled gene by sequencing.

Comparison of Gene Acquisition Methods

MethodSource MaterialKey AdvantagesKey Limitations
DNA ExtractionGenomic DNA from donor organismDirect use of native gene; retains regulatory elementsMay include introns; requires restriction sites
cDNA Synthesis (from mRNA)mRNA of expressed geneIntrons removed; suitable for eukaryotic genesRequires high‑quality RNA; expression level dependent
Chemical SynthesisDesigned nucleotide sequenceCustomisable; no need for donor tissue; can incorporate mutationsCostly for long genes; synthesis errors must be checked

Suggested diagram: Flowchart showing the three pathways (DNA extraction, cDNA synthesis, chemical synthesis) leading to a recombinant gene ready for cloning.

Key Points to Remember

  • All three methods ultimately produce a DNA fragment that can be inserted into a vector for transformation.

  • The choice of method depends on the nature of the gene, the host organism, and the experimental goals.

  • Verification (restriction analysis, PCR, sequencing) is essential regardless of the source.