PCR Flashcards
What is PCR?
Polymerase Chain Reaction
* Uses an enzyme (polymerase) to read a single chain on DNA and get the relevant nucleotides from the environment to make more
* Scaling up the amount of chains so there is enough material to do analysis
What are the steps involved in PCR?
- Start with a double stranded template of DNA
- Heat up to 94°C to denature and helicase split the two strands
- Then hydridise primers at 64°C - add primers to one end of the DNA (3’ to 5’ end)
- Use polymerase, starting on the primer, to attach nucleotides from the environment to artificially replicate DNA at 72°C
- Now we have two double stranded DNA repeats that are exact copies of the template
- Repeat
What can we use PCR to look for?
- Identify genes in <1 week
- Detect mRNAs = gene expression
- Hereditary diseases
- Identify viruses or microbes
- Site-detection mutagenesis
- Paleobiology
- Familial relationships
- Forensic investigations
Why are primers used for PCR?
- polymerase can only add bases to pre-existing strands
- designed to target the edge of STRs (highly variable part of human DNA)
What does primer concentration determine?
the maximum yield of product - used up in each cycle
What are dNTPs?
building blocks of new DNA
What are the 4 dNTPs?
- dATP
- dCTP
- dGTP
- dTTP
How do dNTPs form new bonds?
have an extra OH bond on the deoxyribose sugar
* allows for the bonding to phosphate groups
* builds off the 3’ end of DNA
What is in the buffers when doing PCR?
- salts to stabilise of phosphate groups
- needed for hybridisation and extension
What happens during the initalisation part of the PCR process?
- ensures template DNA is fully suspended and properly denatured
- important if template is long
- hot to activate polymerase
What happens during the denaturation stage of the PCR process?
- splits double stranded DNA into single standed DNA
- higher temp makes DNA more stable in single strands rather than double
What happens during the annealing stage of the PCR process?
- primers bind to template trands
- temperature is lower
- primer binds over complementary template because of high conc
- polymerase will also bind but not proceed yet
What happens during the extension stage of the PCR process?
- synthesis of complementary strand
- temperature is optimised for activity of enzyme
What happens during the cycling of the PCR process?
- each cycle doubles DNA conc
- too few = not enough amplification
- too many = limited by dNTP conc
- too many will lead to truncated products
What happens during the final extension and hold stage of the PCR process?
- ensures all strands are finished
- reduces truncated products
- then cool right down for final hold (most stable temp for DNA so wont degrade) and storing
