Principles of Genetic Technology

· Recombinant DNA = DNA formed by joining DNA from two or more different sources.
· Genetic engineering = deliberate manipulation of genetic material to modify specific characteristics of an organism.
· Genetic engineering may involve transferring a desired gene into an organism so the gene is expressed.
· A transferred gene must be placed where it can be transcribed and translated by the host cell.
· Main uses in this topic: creating recombinant DNA, transferring genes, amplifying DNA, separating DNA fragments, and analysing gene/genome data.

Sources of Genes for Transfer

· Genes inserted into organisms may be obtained by:
· Extracting the gene from donor DNA using enzymes.
· Synthesising DNA from donor mRNA using reverse transcriptase.
· Chemically synthesising DNA from nucleotides when the base sequence is known.
· Using mRNA is useful for eukaryotic genes because mature mRNA has already had introns removed, so the DNA copy contains only exons.
· Chemically synthesised genes allow scientists to design or modify sequences precisely.

Enzymes and Tools Used in Gene Transfer

· Restriction endonucleases cut DNA at specific recognition sequences.
· Restriction enzymes may produce sticky ends, which allow complementary DNA fragments to base-pair.
· DNA ligase joins DNA fragments by forming phosphodiester bonds between sugar-phosphate backbones.
· Plasmids = small, circular DNA molecules used as vectors to transfer genes into host cells, especially bacteria.
· DNA polymerase synthesises new DNA strands by joining complementary nucleotides.
· Reverse transcriptase uses mRNA as a template to make complementary DNA (cDNA).
· A vector must usually contain the desired gene, a promoter, and often a marker gene.

This diagram summarises how a gene of interest is inserted into a plasmid to form recombinant DNA. It links directly to the roles of restriction enzymes, DNA ligase, plasmids, and transformation. Source

Promoters and Gene Expression

· A promoter is a DNA sequence where RNA polymerase binds to start transcription.
· A desired gene may need a promoter transferred with it so the host cell can express the gene.
· Without a suitable promoter, the gene may be present but not transcribed, so no protein is produced.
· The promoter must be recognised by the host organism’s transcription machinery.
· Expression of the inserted gene means the host makes the required mRNA and then the required protein.

Marker Genes and Confirming Gene Expression

· Marker genes help identify cells that have successfully taken up or expressed inserted DNA.
· In this syllabus, marker genes may code for fluorescent products.
· If the marker gene is expressed, transformed cells produce a fluorescent protein and can be identified.
· Fluorescence confirms that the inserted DNA is being transcribed and translated.
· Marker genes are useful because not all host cells successfully take up or express recombinant DNA.

Gene Editing

· Gene editing = a form of genetic engineering that involves insertion, deletion or replacement of DNA at specific sites in the genome.
· It changes DNA at a precise target sequence rather than inserting DNA randomly.
· Gene editing can alter the function of a gene by changing its base sequence.
· Possible changes include removing a faulty sequence, inserting a new sequence, or replacing one allele/sequence with another.
· Exam focus: define gene editing clearly and distinguish it from general gene transfer.

This illustration shows how a gene-editing tool can target a specific DNA sequence and cut it. It is useful for understanding specific-site DNA insertion, deletion or replacement. Source

Polymerase Chain Reaction (PCR)

· PCR = technique used to clone and amplify DNA, producing many copies of a specific DNA sequence.
· PCR requires template DNA, primers, free DNA nucleotides, Taq polymerase, and thermal cycling.
· Taq polymerase is heat-stable, so it does not denature at the high temperatures used in PCR.
· Denaturation: DNA heated to about 95°C; hydrogen bonds break and the double helix separates into single strands.
· Annealing: temperature lowered to about 50–65°C; primers bind to complementary sequences on the template DNA.
· Extension/elongation: temperature raised to about 72°C; Taq polymerase adds nucleotides to primers to form new DNA strands.
· Repeated cycles cause exponential amplification of the target DNA.
· PCR is useful when only a very small DNA sample is available.

This diagram shows the three repeated stages of PCR. It highlights how primers and Taq polymerase allow a target DNA sequence to be copied many times. Source

Gel Electrophoresis

· Gel electrophoresis separates DNA fragments according to length/size.
· DNA samples are placed into wells in an agarose gel.
· An electric current is applied across the gel.
· DNA fragments are negatively charged because of their phosphate groups, so they move towards the positive electrode/anode.
· Shorter DNA fragments move faster and further through the gel than longer fragments.
· A DNA ladder contains fragments of known lengths and is used for comparison.
· Bands can be visualised using stains or fluorescent dyes.
· Gel electrophoresis can be used to compare DNA fragments after PCR or restriction enzyme digestion.

This diagram shows how DNA fragments separate into bands by size. Smaller fragments travel further through the gel, allowing fragment lengths to be compared with a DNA ladder. Source

Microarrays

· Microarrays are used to analyse genomes and detect mRNA in studies of gene expression.
· A microarray contains many short, single-stranded DNA probes fixed in known positions.
· Sample DNA or cDNA made from mRNA is labelled with a fluorescent marker.
· Labelled sample sequences bind to complementary probes by base pairing.
· Fluorescent spots show which sequences are present or which genes are being expressed.
· Stronger fluorescence can indicate a higher amount of matching DNA/cDNA.
· Microarrays allow many genes to be analysed at the same time.

This image represents a DNA microarray, where coloured fluorescent spots show hybridisation between sample nucleic acids and fixed probes. Microarrays are used to compare gene expression across many genes at once. Source

Biological Databases

· Biological databases store information about nucleotide sequences of genes and genomes.
· They also store amino acid sequences of proteins and information about protein structures.
· Databases allow scientists to compare sequences between individuals, species or genes.
· Sequence comparisons can help identify genes, predict protein structure/function, and study relationships between organisms.
· Databases make large-scale genome analysis faster because data can be searched, shared and compared globally.

Common Exam Phrases to Use

· “Restriction endonucleases cut DNA at specific recognition sites.”
· “DNA ligase joins DNA fragments by forming phosphodiester bonds.”
· “A plasmid acts as a vector to transfer the desired gene into a host cell.”
· “A promoter may be required so RNA polymerase can bind and transcription can occur.”
· “PCR amplifies DNA through repeated cycles of denaturation, annealing and extension.”
· “In gel electrophoresis, shorter DNA fragments move further through the gel.”
· “Microarrays detect gene expression by hybridisation of labelled cDNA to complementary DNA probes.”