unit 5 - PCR and gel electrophoresis Flashcards
what is a restriction enzyme
cleaves DNA sequences at sequence specific sites, producing DNA fragments with a known sequence at each end
what is ‘sticky ends’
where the DNA sequence ahs been cut and known base pairs are exposed
what is a primer
a made strand of DNA which binds to the sticky end which signal the polymerase to bind there
what does PCR stand for
polymerase chain reaction
what does PCR do
selectively amplifies particular segment of DNA
what segment is commonly used in PCR
a specific human gene
what is required to complete PCR
starting material ( template DNA)
primers ( 25 base pairs long)
nucleotides
Taq polymerase
MgCl2 / salt buffer
what is Taq polymerase
polymerase from thermally stable bacteria found in a hot pool
why is Taq polymerase used
because it is thermally stable and wont denture at high temperatures
3 stages of PCR
- denaturation
- annealing
- elongation
what is denaturation
separates sequence into single strands by being heated to 95 degrees so it breaks hydrogen bonds
what is annealing
where primers attach to the sticky ends of each original strands at 50-60 degrees
what is elongation
where the strand gets elongated by polymerase adding new complementary nucleotides by forming phosphodiester bonds in the sugar phosphate backbone at 70 degrees
what happens after all 3 steps of PCR are completed
the steps are repeated 40 times
how many copies of DNA is produced by PCR
over a billion
what does PCR do
rapidly amplifies small sections of DNA
what is PCR used for
- disease screening
- DNA finger printing
- species identification
Steps of DNA profiling
1 obtain sample
2 create fragments
3 amplify DNA (PCR)
4 separate fragments (electrophoresis)
5 visualise/ stain fragments
What charge does DNA have
Negative
What end of the tank is DNA attracted too
The positive side
Why is a buffer used
To carry the charge across the tank
How is DNA sorted due to our size
The smaller molecules travel further and faster while the larger ones travel less and slower
What type of current is used in gel electrophoresis
Directional
