Polymerase Chain Reaction - PCR Flashcards
DNA strands are…
…antiparallel
PCR is a method of…
amplifying small amounts of DNA molecules so that we end up with a large number of molecules and can visualise then by gel electrophoresis and used in gene cloning experiments.
What are the uses of PCR?
- forensic DNA analysis – uses microsatellites to detect genetic variation
- amplifying DNA from archaeological specimens – bones
- gene cloning – amplifying genes to place into a plasmid to express a protein/GM
- medical diagnosis
- examining genetic variation in conservation biology
- finding orthologous genes
each strand of the double helix actually is…
…antiparallel to its complementary strand
DNA is polymerised only in…
…one direction
the 5 and 3 here refer to the …
…carbon atoms on the deoxyribose sugar that joins with phosphate groups
DNA polymerase needs …
…small primers to start it of - needs a free 3’ end
Define primer
short sequences of RNA
WHat are primers made by?
made by RNA polymerase
in order to synthesise new strands of DNA several things are needed:
1) a complementary strand of DNA (template strand)
2) short ‘oligonucleotide’ primers ( a free 3’-OH end)
3) molecules of the 4 bases dATP, dCTP, dGTP and dTTP
4) a DNA polymerase enzyme
5) magnesium (Mg2+) ions – a cofactor
6) buffer – correct pH for the enzyme to work
in PCR the aim is to …
…amplify a certain sequence of DNA
if we know some of the sequence of that DNA we can …
…synthesise DNA oligonucleotide primers
if we can then make the DNA strand single stranded we can make …
…the primers pair to the single strands
if we have free nucleotides and a DNA polymerase we can then …
…make a complementary strand
How many steps of PCR?
3
FIrst step of PCR?
Denaturation
Describe denaturation
- By heating the DNA to 95°C we can denature the double strand because the weak hydrogen bonds are broken
- The two strands are separated
Step 2 of PCR?
Annealing
Describe annealing
- bring down the temperature to the annealing temperature of the primers
if we have in excess lots of primer molecules - then those primers will anneal to their complementary sequence
Step 3 of PCR?
Extension
if we now add in a polymerase and the nucleotides dATP, dCTP, dGTP, dTTP then the …
…complementary strand will polymerise
After extension, we have only doubled the strands – we need more to make a useable amount of DNA, so the process …
…goes through another cycle…and another…till we have done ~30 cycles
- as the process is exponential – we end up with huge numbers of strands
most proteins do not like being heated up – they …
…denature
some bacteria live at …
…very hot temperatures (in hot springs for example
we use heat stable polymerases isolated from …
…the thermophilic bacteria Thermus aquaticus – Taq polymerase
because the polymerase is heat stable we can …
… continuously cycle the PCR reaction between 3 temperatures
What are the three temperatures PCR can be cycled between?
1) denaturation temperature
2) annealing temperature
3) extension temperature
WHats the denaturation temperature?
95 deg c (takes 1 minutes)
Whats the annealing temperature?
40-65°C usually (takes 1 minute)
WHats the extension temperature?
72 deg C (time depends on the length of DNA being amplified)
Why is extension temperature 72 deg C?
the optimum temperature at which Taq works
results of PCR can be seen on …
…gel electrophoresis
the first lane is a …
…ladder
What are ladders in gel electrophoresis?
Size markers
The second lane is…
…a negative control
What is a negative control?
no DNA added to the PCR reaction
Lanes 3 and 4 are from…
…whitefly DNA and both have the virus
How many lanes in gel electrophoresis?
4 lanes
Why do proteins denature at high temp?
Changes structure
What are the reactants used in PCR?
target DNA, primers, polymerase and nucleotides
WHat is done with the reactants in PCR?
pipetted into a PCR tube
After the reactants are pipetted into a PCR tube, the tubes are then placed into a…
thermal cycler
Function of the thermal cycler used in PCR?
which heats the tubes up to the correct temperature for the correct amount of time and ‘cycles’
the design of primers is very important and several things need to be taken into consideration:
1) obviously the sequence has to be complementary
2) length of primer – usually around 20-25 bases (the longer it is the higher the annealing temperature and the more specific it is)
3) at least 50% of the bases should be G or C (because they have more hydrogen bonds – they increase the annealing temperature)
4) there should not be repeats within the bases
5) the primers shouldn’t be complementary to each other
6) if possible the 3’ (last) base should be a G or C
all living organisms have …
…DNA genomes
In many cases DNA genomes of different organisms have…
…different versions of the same genes – express the same or similar proteins which do the same job in the cell – these are called orthologues
Orthologues are derived from….
…same gene but are only different as they have separated due to a speciation event
orthologous genes are …
…conserved
the closer that species are related to each other (through evolution) the more …
…their DNA sequences are similar
