19.1 Principles of Genetic Technology

1. Select DNA sequences from the sources that provide the required information.
2. Combine the sequences into one DNA construct; the construct is recombinant DNA.
3. If the construct is introduced into a host cell, it may be copied and/or expressed there when the host has the required cellular control.
4. A host containing introduced genetic material is a genetically modified organism (GMO); if the introduced sequence came from another species, it is also described as transgenic.

1. Identify the gene associated with the desired characteristic.
2. Isolate or obtain that gene as a suitable DNA sequence.
3. Cut the relevant DNA and the delivery construct, then insert and join the gene to form recombinant DNA.
4. Introduce the recombinant DNA into a host cell using a delivery system.
5. Select or identify cells that received the construct, then clone the successful cells when required.
6. Check that the transferred gene is expressed and that the intended product or characteristic is obtained.

Use the source-selection method:

1. Define the required characteristic and the nucleotide sequence that encodes it.
2. Choose a source: extract the sequence from donor DNA; use specialised-cell mRNA as a template for cDNA; or design and synthesise the sequence from the genetic code.
3. Obtain the desired sequence: the donor-DNA route cuts it from the genome, the mRNA route makes single-stranded cDNA and then double-stranded DNA, and the synthesis route joins short DNA pieces into a longer sequence.
4. Check that the product contains the required gene sequence before handing it to the later DNA-transfer step.

Genomic DNA — directly represents a donor chromosome, but may include introns and other non-coding DNA.
cDNA from mRNA — reflects a gene that was expressed in the donor cell and lacks introns present in the processed mRNA.
Artificial synthesis — uses known genetic information to design the sequence, without cutting it from an existing genome; it can avoid introns or unnecessary non-coding sections.

Promoter choice can control where, when and how strongly transcription occurs. A constitutive promoter can keep expression active, while a regulated promoter can respond to a condition or restrict expression to particular tissues when the construct and host support this.

1. Add the marker gene to the same plasmid or construct as the gene of interest.
2. Place both under the same promoter or linked expression control so they are transcribed together.
3. Transfer the construct into host cells and allow transcription and translation.
4. Expose the cells to the appropriate ultraviolet or blue light. Cells producing the fluorescent protein glow, identifying cells in which the linked transferred genes are being expressed.

The reaction needs target DNA, forward and reverse primers, free nucleotides, a DNA polymerase, and a buffer. Primers are complementary to the ends of the target region, so they provide specificity and tell the polymerase where to begin. Taq polymerase is useful because it remains active after the high-temperature denaturation stage.

1. Denaturation: heat the double-stranded template so hydrogen bonds break and the strands separate (about 95°C in the SME method).
2. Annealing: lower the temperature so the forward and reverse primers bind to complementary sequences at the target ends (about 50–60°C).
3. Extension: raise the temperature to the polymerase optimum so complementary strands are built from free nucleotides (about 72°C for Taq).
4. Repeat the cycle: each cycle doubles the target DNA, producing exponential amplification of the region between the primers.

1. Prepare the DNA sample and an agarose gel with wells, then submerge the gel in electrolyte solution.
2. Load the DNA fragments into the wells and position the wells at the negative-electrode side.
3. Apply the electric field: negatively charged DNA migrates through the gel towards the positive electrode.
4. Separate the fragments: smaller or shorter fragments move more easily through the pores and therefore travel faster and farther than larger fragments.
5. Make the separated DNA visible using an appropriate stain, fluorescent label or labelled-probe detection method supported by the investigation.
6. Compare the band pattern with a size ladder and suitable control fragments to estimate sizes or assess similarity.
7. Interpret cautiously: a matching pattern supports similarity under the tested preparation and conditions, but band evidence alone does not establish absolute identity.

Direction is set by DNA charge, while separation distance is influenced by fragment length and the gel conditions. A ladder or control provides the reference for comparison; distance is a conditional size estimate, and band brightness is not itself a fragment-length measurement.

1. Choose probes with sequences complementary to the genes or transcripts being investigated and fix the probes at known spots on the chip.
2. Collect the sample material. For a gene-expression comparison, collect mRNA and use reverse transcriptase to make corresponding cDNA; fluorescently label the sample nucleic acids.
3. Denature the labelled material where needed so complementary single strands can pair with the probes.
4. Allow the sample to hybridise with the probes on the chip, then wash away material that has not hybridised.
5. Scan the chip using the supported fluorescence or ultraviolet detection method. A coloured spot indicates hybridisation to the corresponding probe.
6. Compare spot colour or presence with the relevant sample, control and background. Presence can support sequence or transcript detection; for expression studies, relative spot intensity indicates relative mRNA abundance after appropriate comparison and normalisation.
7. State the conclusion with its evidence boundary: a signal reports probe binding under the test conditions, not automatically a complete gene sequence, protein amount or biological function.

Sequence-presence reading — a coloured spot supports that a complementary sequence was detected.
Expression reading — stronger or weaker fluorescence is interpreted comparatively as more or less labelled cDNA from mRNA, provided samples were prepared and normalised comparably.
Controls and background — use matched controls and account for background signal before comparing spots; no probe number, intensity threshold or universal cut-off is assumed.