PCR Flashcards
what does pcr stand for
polymerase chain reaction
what does pcr do
amplifies the size of a DNA sample by making a large number of identical copies of a piece of DNA
examples of places you can obtain these DNA tissue samples
- crime scenes
- archeological specimens
- dead/living relatives
(good for DNA fingerprinting)
what do you have to do before starting step 1 of pcr
cut out the chromosome carrying the gene into fragments using restriction enzymes
then you can put the sample into the PCR machine
Step 1 (PCR)
DENATURATION
the fragments is heated to break the hydrogen bonds which separates the two halves of the DNA ladder into two separate strands
@ 95*C
Step 2 (PCR)
ANNEALING
a large quantity of single-stranded primer sequences is added. The solution is cooled and these primers attachthemselves to the ssDNA at one end of each DNA piece
@ 50*C temperature
ssDNA: single strande DNA
Step 3 (PCR)
EXTENSION
thermophilic DNA polymerase is added to the mixture and as the temoerature is raised this enzyme synthesizes double-stranded DNA from the starting point of the primer on each ssDNA piece
@ 72*C
where does the name thermophilic come from
thermophile: likes hot temps
comes from “Thermus aquaticus”
its a bacteria that lives in hot springs
aka taq
Step PHO (PCR)
REPEAT
now there are two copies of the original DNA fragment. the cycle is repeated (steps 2-4) and then there are 4 fragments. repeating the cycle agains gives 8 fragments, again for 16, 32, 64, 128, 256, 512, 1024, etc
within hours there can be millions of identical copies of the original DNA fragments
why do you have to be cautious during PCR
want to avoid any possible contamination from the DNA of the person taking the sample or other stray pieces of DNA in the surroundings . any of the contamination DNA will be amplified along with the target DNA
what you need for PCR
- DNA sample
- taq polymerase
- PCR machine
- nucleotide
- primer
