Genetics And Gene Expression Flashcards
(25 cards)
Where Transcription
Takes place in the nucleus of eukaryotic cells.
Transcription
- Initiation:
• RNA polymerase binds to the promoter region of the DNA.
• The DNA strands unwind and separate at the gene to be transcribed.- Elongation:
• RNA polymerase moves along the template (antisense) DNA strand.
• It synthesises a single-stranded pre-mRNA molecule by adding complementary RNA nucleotides (A, U, C, G) in the 5’ to 3’ direction.
• Uracil (U) replaces thymine (T). - Termination:
• RNA polymerase reaches a termination sequence.
• Pre-mRNA detaches from the DNA template.
• DNA strands rewind back together. - Processing (eukaryotes only):
• Pre-mRNA undergoes splicing to remove introns.
• A 5’ cap and poly-A tail are added.
• The mature mRNA exits the nucleus through nuclear pores.
- Elongation:
RNA Splicing
- The initial pre-mRNA contains exons (coding sequences) and introns (non-coding sequences).
- Spliceosomes (complexes of proteins and small nuclear RNAs) recognise specific sequences at the intron-exon boundaries.
- The spliceosome cuts out the introns and joins the exons together.
- The result is a mature mRNA molecule containing only exons, which can be translated into a protein.
Where translation
Occurs in the cytoplasm on ribosomes (either free or on the rough ER).
Translation
- Initiation:
• The small ribosomal subunit binds to the mRNA at the start codon (AUG).
• A tRNA with the complementary anticodon (UAC) carrying methionine binds to the start codon.
• The large ribosomal subunit attaches, forming a complete ribosome.- Elongation:
• tRNAs bring specific amino acids to the ribosome according to the codons on the mRNA.
• Each tRNA has an anticodon complementary to the mRNA codon.
• Amino acids are linked by peptide bonds via the enzyme peptidyl transferase.
• The ribosome moves along the mRNA in the 5’ to 3’ direction. - Termination:
• When a stop codon (UAA, UAG, UGA) is reached, translation ends.
• The polypeptide chain is released.
• Ribosome subunits separate.
- Elongation:
- Substitution (point mutation):
• One base in the DNA is swapped for another.
• Effects:
• Silent mutation: New codon codes for the same amino acid — no change in protein.
• Missense mutation: New codon codes for a different amino acid — may alter protein function.
• Nonsense mutation: New codon becomes a stop codon — leads to premature termination of protein.
- Deletion (frameshift mutation):
• One or more bases are removed from the DNA sequence.
• Shifts the reading frame of codons downstream.
• Usually results in completely different amino acids and often a nonfunctional protein.
• Can cause premature stop codons.
- Insertion (frameshift mutation):
• One or more bases are added into the DNA sequence.
• Also shifts the reading frame downstream.
• Same effects as deletion — usually disastrous for protein function.
Epigenetic Regulation
What is it?
• Control of gene expression without changing the DNA sequence.
• Changes how genes are turned on or off.
- DNA methylation
• Addition of methyl groups (–CH₃) to cytosine bases in DNA, often near gene promoters.
• Methylation usually silences genes by preventing transcription.
- Histone modification
• Chemical groups (e.g., acetyl, methyl) added to histone proteins around which DNA is wrapped.
• Changes how tightly DNA is wound.
• Acetylation usually loosens chromatin, promoting transcription.
• Deacetylation tightens chromatin, reducing transcription.
Epigenetic regulation Effects:
• Epigenetic changes can be stable and heritable but reversible.
• Allow cells to specialise by turning certain genes on/off.
• Environmental factors (diet, stress) can influence epigenetics.
What is siRNA
• Small interfering RNA (siRNA) is a short, double-stranded RNA molecule involved in RNA interference (RNAi).
• It regulates gene expression by silencing specific mRNA molecules.
How does siRNA work
- Formation of siRNA:
• Long double-stranded RNA molecules are cut into siRNA fragments by an enzyme called Dicer.- Incorporation into RISC:
• The siRNA unwinds, and one strand (the guide strand) is incorporated into the RNA-induced silencing complex (RISC). - Targeting mRNA:
• The siRNA within RISC binds complementary sequences on target mRNA. - mRNA degradation:
• RISC cleaves the target mRNA, causing it to be degraded and preventing translation.
- Incorporation into RISC:
Result of siRNA
• Gene expression is silenced because the mRNA is destroyed before it can be translated into protein.
- Restriction Enzymes (Restriction Endonucleases) in recombinant DNA
• These are enzymes that cut DNA at specific sequences called recognition sites.
• Recognition sites are usually palindromic sequences (same forwards and backwards).
• They can make:
• Sticky ends (overhanging single-stranded ends), or
• Blunt ends (straight cuts).
• By cutting both the DNA you want to insert (e.g., a gene) and the plasmid vector with the same restriction enzyme, you get matching sticky ends that can pair up.
- DNA Ligase in recombinant DNA
• This enzyme joins DNA fragments together by forming phosphodiester bonds between sugar-phosphate backbones.
• After the sticky ends from the plasmid and the gene align by base pairing, ligase seals the sugar-phosphate backbone, making a stable recombinant DNA molecule.
- Why It restriction enzymes and ligase matter for recombinant DNA
• Restriction enzymes allow scientists to cut DNA at precise locations.
• DNA ligase allows these fragments to be joined into vectors, like plasmids, for cloning or gene expression.
• This is fundamental for genetic engineering — like making GM crops, gene therapy, or cloning genes.
PCR (Polymerase Chain Reaction)
Purpose:
• To amplify (make many copies of) a specific DNA sequence quickly.
Main stages of PCR:
- Denaturation (about 95°C):
• The double-stranded DNA heats up and separates into single strands by breaking hydrogen bonds.- Annealing (about 50–65°C):
• The temperature lowers so primers (short DNA sequences complementary to target DNA ends) can bind (anneal) to their specific sequences on the single-stranded DNA. - Extension (about 72°C):
• Taq DNA polymerase extends the primers by adding complementary nucleotides in the 5’ to 3’ direction, synthesising new DNA strands.
- Annealing (about 50–65°C):
Cycle repetition:
• These 3 steps repeat 25–35 times, doubling the DNA amount each cycle — exponential amplification.
Gel Electrophoresis
Purpose:
• To separate DNA fragments based on their size.
Gel electrophoresis method
- Preparation:
• DNA fragments are placed into wells in an agarose gel.
• The gel is submerged in a buffer solution that conducts electricity.- Electric current:
• A voltage is applied across the gel.
• DNA fragments, which are negatively charged (due to phosphate groups), migrate towards the positive electrode (anode). - Separation by size:
• Smaller DNA fragments move faster and further through the gel pores.
• Larger fragments move slower and less far. - Visualisation:
• DNA is stained with a dye (like ethidium bromide or safer alternatives).
• Under UV light, the DNA fragments show up as bands.
• The distance travelled by fragments can be compared to a DNA ladder (marker) to estimate fragment sizes.
- Electric current:
Genetic Fingerprinting – AQA A-Level Biology
What is it?
• A technique used to identify individuals based on unique patterns in their DNA.
Genetic fingerprinting method
- DNA extraction:
• DNA is extracted from cells (e.g., blood, hair).- DNA cutting:
• DNA is cut into fragments using restriction enzymes at specific sequences. - PCR amplification (optional):
• Specific regions of DNA containing Variable Number Tandem Repeats (VNTRs) or Short Tandem Repeats (STRs) are amplified using PCR. - Gel electrophoresis:
• The fragments are separated by size using gel electrophoresis, producing a pattern of bands. - Visualisation:
• DNA is stained and viewed under UV light, creating a DNA fingerprint unique to the individual (except identical twins).
- DNA cutting:
