4c- PCR - polymerase chain reaction Flashcards
What is polymerase chain reaction (PCR)?
A laboratory technique used to produce many identical copies of DNA from a small initial sample.
What does it mean to amplify?
To increase the quantity of a molecule by making many copies.
What is a primer?
A short, single strand of nucleic acids that acts as a starting point for polymerase enzymes to attach.
What is a restriction endonuclease?
Any enzyme that acts like molecular scissors to cut nucleic acid strands at specific recognition sites.
What does it mean to denature?
The disruption of a molecule’s structure by an external factor such as heat.
What is Taq polymerase?
A heat-resistant DNA polymerase enzyme isolated from the bacteria Thermus aquaticus, which amplifies a single-stranded DNA molecule by attaching complementary nucleotides.
What does it mean to elongate?
To synthesise a longer polynucleotide.
What is a thermal cycler?
A laboratory apparatus which alters the temperature in pre-programmed steps for temperature-sensitive reactions like PCR.
What does it mean to anneal?
The joining of two molecules, for example two complementary DNA strands during the cooling phase of PCR.
What is a forward primer?
A DNA primer that binds to the 3’ end of the template strand and reads the DNA in the same direction as RNA polymerase.
What is a reverse primer?
A DNA primer that binds to the 3’ end of the coding strand and reads the DNA in the reverse direction to RNA polymerase.
process of PCR
1- Denaturation – DNA is heated to approx 90–95 °C to break the hydrogen bonds between the bases and separate the strands, form single-stranded DNA.
2 -Annealing – the single-stranded DNA is cooled to approximately 50–55 °C to allow primers to bind to complementary sequences on single-stranded DNA.
3 -Elongation – the DNA is heated to 72 °C, allows Taq polymerase to work optimally. Taq polymerase binds to the primer (acts as a starting point) and begins synthesising a new complementary strand of DNA.
4 Repeat – the cycle (steps 1–3) is repeated multiple times to create more copies of DNA.
