Transcription and Translation

• Transcription uses a DNA sequence as a template to produce a complementary mRNA sequence.

Transcription steps

• Begins with initiation, where RNA polymerase binds to the promoter region with the help of transcription factors, allowing transcription to start.
• Elongation involves RNA polymerase reading the DNA template strand to produce pre-mRNA.
• Termination occurs when RNA polymerase reaches a termination sequence, detaching and releasing the pre-mRNA sequence.

Post-transcription

• Pre-mRNA undergoes post-transcriptional modification before it exits the nucleus and moves to the ribosome.

Translation

• Translation uses mRNA sequence to produce a corresponding amino acid sequence (polypeptide).
• Initiation starts when the 5' end of mRNA binds to the ribosome, and the start codon (AUG) is recognized, signalling the beginning of translation.
• Elongation continues as the ribosome reads the mRNA, bringing required tRNA (amino acids), which bind to adjacent amino acids with a peptide bond through a condensation reaction.
• Termination continues until a STOP codon is reached; this signals the end of translation, as there are no corresponding tRNA molecules for stop codons.

The Genetic Code

• It's the set of rules by which information is encoded in genetic material.
• "Coded information" refers to the nucleotide sequences in DNA template strands.
• "Decoded information" refers to the amino acid order in polypeptides.
• The genetic code is used to construct the correct amino acids in the correct sequence to create polypeptides.

Eukaryotic Gene Expression

• Eukaryotic gene expression involves DNA transcription and protein translation.
• Gene expression regulation can occur through activation & repression.
• A promoter is a DNA sequence where RNA polymerase binds.
• A leader region is the segment of DNA or mRNA immediately before the coding region, involved in gene regulation.
• A TATA box is a type of promoter region in eukaryotic cells, with the base sequence 'TATAAA'.
• Introns are non-coding DNA regions spliced out during RNA processing.
• Exons are DNA regions that code for proteins and are not spliced out during RNA processing.
• A termination sequence is a DNA sequence that signals the end of transcription.

Protein Structure

• Primary structure is the amino acid sequence assembled into a polypeptide chain on the ribosomes.
• Linear sequence is determined by genetic material in nucleus
• Secondary structure occurs as amino acid chains form 3 different folding patterns, determined by the R groups of the amino acids.
• Alpha helix is a spiral molecule held together by hydrogen bonds between neighboring CO & NH groups.
• Beta-pleated sheets are folds in the polypeptide chain, formed by hydrogen bonds.
• Structure can also exist in random coils - shape that is neither alpha or beta.
• Tertiary structure involves the folding of random coils, alpha helices, and beta sheets.
• Various interactions between the R groups of amino acid residues determine the tertiary structure.
• Tertiary folding results in a permanent functional shape or conformation.
• Quaternary structure describes the complex patterns that are made up of more than one polypeptide chain which can be same or different.
• Held together by H and disulphide bonds

Restriction Enzymes

• Molecular scissors that cut DNA at specific recognition sites.
• Base sequences at which endonucleases cut are palindromic which means complementary read backwards.
• Restriction enzymes are naturally produced by bacteria as a defense against viruses and DNA is cut at precise places.
• Restriction enzymes cut DNA at specific sites by breaking the bonds in a polynucleotide chain.
• Each enzyme cuts DNA at certain points, restricted to specific locations.
• Enzyme's cutting positions are its recognition sequence/site, with a specific nucleotide order.

Ligases

• Molecular glue, they function in the opposite direction to endonucleases.
• Ligases join DNA or RNA together, catalyzing the formation of phosphodiester bonds to merge fragments.
• They are not specific and can join both blunt and sticky end pieces, potentially producing one longer DNA piece.

Polymerases

• Polymerases synthesize polymer chains from monomers.
• RNA polymerase is used in transcription, and DNA polymerase in replication or amplification.
• DNA amplification requires primers, short single-stranded nucleic acid pieces that start polymerase attachment.
• Synthesis occurs in a 5' to 3' direction.

CRISPR-Cas9

• A gene editing system naturally found in bacteria and used to edit genes in other organisms.
• In bacteria plays an important role in defense against viral attacks (bacteriophage)
• Cas9 is an enzyme that when attached to gRNA can cut a target sequence of DNA.
• gRNA (guided RNA) has a specific sequence determined to guide Cas9 to a specific site.
• Spacers are short DNA sequences from invading bacteriophages added into the CRISPR sequence.
• PAM (protospacer adjacent motif) is a 2-6 nucleotide sequence next to the DNA targeted by Cas9, allowing bacteria to differentiate between self & non-self.
• Synthetic gRNA is created in a lab with a complementary spacer to the target DNA.
• Cas9 enzyme is obtained with an appropriate target Protospacer Adjacent Motif or PAM (sequence of 2-6 nucleotides found next to the DNA targeted by Cas 9), e.g. PAM site contains the sequence NGG.
• Cas9 and gRNA are added together in a mixture and bind together to create the CRISPR-Cas9 complex which is then injected into a specific cell, such as a zygote.
• The Cas9 finds the target PAM sequence and checks whether the gRNA aligns with the DNA, following which Cas9 cuts the selected sequence of DNA giving the DNA a blunt end cut that the cell will attempt to repair.
• When repairing the DNA, the cell may introduce new nucleotides into the DNA at this site. Scientists may inject particular nucleotide sequences into the cell with the hope that it will ligate into the gap.

Applications of CRISPR-Cas9

• Can cure or treat a variety of genetic disorders.
• Can 'knock out' genes to identify their functions.
• Can snip out a faulty gene segment and replace it with a working copy.
• Can activate or repress genes, add a new gene to the genome, or advance animal welfare (e.g., hornless dairy cows).
• Can modify crops to increase nutritional value & genetically modified food.
• Can locate certain genes by attaching fluorescent proteins to Cas9.
• Is versatile in that it can be programmed to target any specific sequence dictated by gRNA whereas restriction endonucleases will only act on a single restriction site, which cannot be programmed

Polymerase Chain Reaction

• Artificial form of DNA replication.
• A multistep process that amplifies DNA and uses multiple materials.
• A DNA sample, and a polymerase enzyme called Taq polymerase are required
• Taq polymerases are enzymes found in bacteria that can withstand high heat temperatures and can be used to synthesise DNA or RNA.
• Free Nucleotide bases (A, T, C, G) and DNA primers (bind to the single stranded DNA and provide a starting point for DNA synthesis) are needed
• A Buffer solution (to provide a suitable chemical environment for Taq by maintaining the correct pH) is needed
• Trace amounts of DNA need to be amplified to millions of copies so that there is enough DNA for further investigation. (PCR)
• PCR STEP 1: DENATURATION; Denaturing the DNA (90 - 95°C)
• At this temperature the hydrogen bonds between the two strands of DNA are broken resulting in two separate strands of DNA
• PCR STEP 2: ANNEALING OF PRIMERS Primer annealing (50 - 55°C) (temp can vary)
• The temperature is lowered to allow the primers to bind (anneal) to their complementary bases on each of the single strands of DNA. If temp is too low, double strands of DNA reform.
• PCR STEP 3: EXTENSION: DNA synthesis (72°C)
• Taq DNA polymerase extends the DNA strand from the primers using the base pairing rule (in different directions – anti-parallel).
• CYCLE REPEATED - EXPONENTIAL AMPLIFICATION

Gel electrophoresis

• Used in sorting DNA fragments, including the interpretation of gel runs for DNA profiling
• Gel electrophoresis separates DNA fragments by length for analysis.
• When DNA fragments are cut with different restriction enzymes, fragments of different sizes are formed.
• The DNA samples are placed into wells at one end of a thin slab of agarose gel.
• An electric current is passed through the gel to sort the fragments.
• Each nucleotide in a DNA molecule contains a negatively-charged phosphate group, so DNA is attracted to the positive electrode.
• The molecules diffuse through the gel, and smaller lengths of DNA move faster than larger lengths.
• Dye is added to track the DNA as DNA fragments are invisible
• When the electric current is turned off a fluorescent dye (e.g. ethidium bromide) is then used to stain the DNA fragments so that the DNA becomes visible.
• Each band of DNA may contain many millions of fragments of the same size. The smaller the length of a DNA molecule, the further down/along the gel it will move in a given time.
• Hence wells are closest to the negative electrode