Biomolecular Processes: PCR & Gene structure

• DNA polymerase adds nucleotides to extend the primers, building new DNA strands.
• These steps are repeated to double the amount of target DNA each cycle.
• Molecular principle mimics natural DNA replication in a test tube.
• Specific primers target only the desired region.
• Heat-stable polymerase survives high temperatures.
• Exponential amplification occurs.

Gene Structure and Expression

• A gene is a segment of DNA that carries the instructions to make a protein or RNA molecule.
• Promoter is the "start" signal for transcription, where RNA polymerase binds.
• Exons are coding regions that contain instructions for making proteins.
• Introns are non-coding regions that are removed later.
• Terminator signals the end of transcription.
• Gene expression has two steps: transcription and translation.

Transcription

• Occurs in the nucleus.
• RNA polymerase lands on the promoter region of a gene, signaling the start.
• The double helix unwinds, exposing the template strand.
• One strand (antisense) acts as a template.
• RNA polymerase moves along the template strand, adding complementary ribonucleotide units to the 3' hydroxyl end.
• Sequences called promoters, such as the TATAAA box, are where RNA polymerase lands.

mRNA Processing

• Introns are removed, and exons are spliced together.
• This creates functional mRNA for translation into protein.
• 5' processing involves capping with methylated guanine.
• 3' processing involves cleavage and polyadenylation.

DNA Replication

• DNA replication is the process by which a cell makes an exact copy of its DNA before cell division, ensuring each new cell gets a complete genome.
• Enzyme helicase unwinds the double helix by breaking the hydrogen bonds between base pairs.
• This creates a replication fork.

Priming

• Primase (enzyme) adds a short RNA primer to give DNA polymerase a starting point.

Elongation

• DNA polymerase adds new nucleotides to the 3' end of the growing strand, following base pairing rules.
• One strand (leading) is made continuously, and the other (lagging) is made in short pieces called Okazaki fragments.
• DNA polymerase I joins any gaps.
• Ligase joins Okazaki fragments.
• Semiconservative process means one strand is old and one strand is new in each new DNA molecule.

Proofreading

• DNA polymerase can detect the insertion of an incorrect base and backs up to remove the mistakenly inserted base.
• DNA polymerase uses exonuclease activity to remove the incorrect base and replace it.
• Base pair geometry aids in the recognition of incorrect pairs.
• Active sites know if pairing deviates from appropriate geometry.

Translation

• Translation occurs in the cytoplasm.
• It's the process by which the genetic code carried by mRNA is decoded to synthesize proteins.
• Ribosomes read the mRNA in codons (3-letter sequences).
• tRNA molecules bring amino acids that match each codon.
• The ribosome links amino acids into a polypeptide chain.
• The chain folds into a functional protein.
• AUG, is the start codon and codes for methionine.
• UAG/UGA are termination codons.

Polymerase Chain Reaction (PCR)

• PCR is a lab technique used to amplify (copy) a specific segment of DNA in vitro (outside the body) using enzymes and temperature cycles.

PCR Components

• Target DNA contains the target sequence you want to amplify.
• Primers are short, single-stranded DNA pieces that are complementary to the start and end of the target sequence.
• DNA polymerase is an enzyme that builds new DNA strands from nucleotides.
• dNTPs (dATP, dCTP, dGTP, dTTP) are nucleotides used to build DNA strands.
• Buffer maintains optimal pH and salt conditions for the reaction.

PCR Steps

• Double DNA strands are denatured by heating to break hydrogen bonds.
• Separated strands are cooled so primers bind to their complementary sequences on each single strand.