MG (PCR) Flashcards
What’s PCR?
- Polymerase Chain Reaction
- method of copying fragments of DNA.
- It is an automated method (rapid and efficient).
What things are needed for PCR?
- Small DNA sample
- Taq DNA polymerase
- Primers
- Nucleotides
- Thermo cycler
What are the 3 steps in PCR
- denaturation (95C)
- annealing (68C)
- elongation/extension (72C)
Outline denaturation
- DNA fragments (sample), primers, DNA polymerase and free nucleotides added to a thermocycler.
- 94-96°C temperatures cause DNA strands to separate (denature) – hydrogen bonds are broken.
Outline annealing
- Primers anneal (join) to their complimentary bases (68°C).
- This provides a starting point for DNA polymerase.
Outline elongation
- Temperature increased to 72°C, the optimum temperature of DNA polymerase.
- DNA polymerase attaches free nucleotides to each of the separated DNA template strands.
- The cycle re-starts and the DNA will be amplified (double each time).
PCR in more detail…
- The sample of DNA is mixed with DNA nucleotides, primers and Taq DNA polymerase.
- The mixture is heated to around 95C to break the H bonds between complementary base pairs and thus denature the double-stranded DNA into two-single strands of DNA.
- Mixture is cooled to around 68oC so that the primers can anneal to one end of each single strand of DNA. This gives a small section of double stranded DNA at the end of each single-stranded molecule.
- Taq DNA polymerase binds to the end where its double stranded, 72oc is optimum temperature.
- Temp is raised to 72oC and this keeps the DNA as single strands.
- Taq polymerase catalyses the addition of DNA nucleotides to the single-stranded DNA molecules, starting at the end with the primer and proceeding in the 5’ to 3’ direction.
- When Taq DNA polymerase reaches the other end of the DNA molecules then a new double strand of DNA has been generated.
- The whole process begins again and is repeated for many cycles.
- The amount of DNA increases exponentially 1,2,4,8,16,32,64,128 and so on.
Advantages of PCR (in vitro cloning )
- Very rapid
- Does not require living cells
State some applications of PCR
- Detection of oncogenes
- Detecting mutations
- Forensic science
- Tissue typing
- Monitoring the spread of infectious disease
- Identify viral infections
- Research
How can PCR be useful in forensic science?
Small quantities of DNA can be amplified to identify criminals or ascertain parentage
How can PCR be useful in tissue typing?
Donor and recipient tissues can be matched to reduce the risk of rejection
How can PCR be useful in detection of oncogenes?
Detect the type of mutation leading to cancer. Can inform medication
PCR in terms of detecting mutations?
Detecting genetic diseases. Parents may do this before conceiving
How can PCR be useful in identifying viral infections?
Verify the type of viral infection present
How can PCR be useful in monitoring the spread of infectious disease?
helps in monitoring the emergence of new strains
