PCR Flashcards
PCR stands for _____
Polymerase Chain Reaction
PCR is a technique which allows . . .
The amplification of one DNA strand into millions of copies.
PCR has had a major impact on ____
Disease diagnosis, medicinal research and forensic investigation
PCR is basically a form of ____ for use in the laboratory. The technique simply provides ____ and automates the process.
PCR is basically a form of DNA replication for use in the laboratory. The technique simply provides the conditions and materials necessary for DNA replication and automates the process.
The 4 main requirements for PCR are
- Template Strands
- RNA Primer
- DNA Nucleotide triphosphates
- Thermostable DNA Polymerase enzymes
Name the 3 steps of PCR
- Denaturation - (Seperation of the strands)
- Annealment - (Attachment of primers)
- Elongation - (Formation of new DNA strands)
Describe the first step of PCR
The double-stranded DNA is denatured into single strands by heating to 95 C for a few seconds. This is a high enough temperature to break the hydrogen bonds between the complimentary nitrogenous bases between the antiparallel strands.
Describe the second step of PCR
The solution containing the now separated strands of DNA is cooled to 55 ºC to allow specific primers made of DNA to anneal (join) to a complementary sequence on the template DNA strands. During replication, the entire DNA strand is copied. In PCR, only one or two genes are usually amplified, therefore DNA primers are designed that anneal to sites close to the 3’ end of the required gene.
Describe the thrid step of PCR
Because the optimum temperature of the thermostable DNA polymerase is higher than human DNA polymerase, the solution is now heated slightly to 72 ºC to allow the DNA polymerase to extend the primers by adding DNA nucleotides to the 3’ ends. This extends the newly forming DNA strands in the 5’ to 3’ direction.
Number of DNA molecules after X cycles =
Number of original DNA molecules x 2^number of cycles
