Untitled Quiz

Study Notes

PCR is an in-vitro enzymatic DNA amplification technique used to make millions of copies of a specific DNA sequence in a few hours.
PCR requires knowing the DNA sequence to be amplified or at least the sequences flanking the target region.
PCR can be used to amplify DNA from various sources: viral, bacterial, plant, and animal.

Genome mapping and gene function determination.
Biodiversity studies (e.g., evolution studies).
Detection of drug resistance genes.
Forensic analysis of DNA samples (e.g., paternity test).
DNA fingerprinting for analyzing evidence from crime scenes using a single hair or spot of blood.
Diagnostics: Prenatal testing of genetic diseases, early detection of cancer, diagnosis of viral infections (Hepatitis B, C, AIDS, COVID-19), and diagnosis of genetic disorders (e.g., sickle cell anemia).

Template DNA: The sample of DNA that you wish to replicate.
Two Primers (forward and reverse): These act as starting/ending points for DNA replication, defining the region of DNA to be replicated. Primers are typically 15 to 30 nucleotides long, synthetically produced, and complementary to the 3' ends of the target DNA.
DNA Polymerase: The enzyme that replicates DNA. Taq DNA polymerase is commonly used, a thermostable enzyme derived from the bacterium Thermus aquaticus.
dNTPs (Deoxyribonucleoside Triphosphates): (dATP, dCTP, dGTP, dTTP) are the building blocks for new DNA strands.
Mg2+: An essential cofactor for DNA polymerase.

Denaturation: The target DNA is heated to 95°C to separate double-stranded DNA into single strands.
Annealing: The reaction is cooled to 50°C, enabling the two primers to anneal (bind) to their complementary sequences on the single-stranded DNA.
Extension: The temperature is raised to 72°C, allowing DNA polymerase to extend the primers, synthesizing new DNA strands complementary to the original ones, using dNTPs.

A programmable device called a thermocycler is used to cycle between specific temperatures for set periods of time during PCR.
Each PCR run commonly consists of 20 to 30 cycles, exponentially amplifying the DNA by a million to billion fold.
The thermostable Taq polymerase allows for high temperatures without losing activity.

The resulting PCR products are separated by gel electrophoresis and visualized under a UV transilluminator.

PCR requires a heat-stable DNA polymerase to withstand the high temperatures involved in the reaction.
Each PCR cycle doubles the amount of DNA.
The resulting PCR products can be used for various applications, including diagnostic testing, gene sequencing, and forensic analysis.