PCR, gel electrophoresis, recombinant plasmids #4 Flashcards
enzymes used to manipulate DNA
- polymerase
- ligase
- endonucleases
polymerase chain reaction - PCR
a technique used to make multiple copies of specific sections of DNA.
- DNA amplification
- useful if the original DNA is limited in quality (crime scenes, extinct species, viral infections)
stages of PCR
- denature
- anneal
- extension
denature
keep the DNA at 95C for 2 minutes to separate the double stranded DNA into single strands
anneal
the DNA is held at 55C for 2 minutes while short single strand of DNA (primers) bind to the denatured DNA by base pairing at either end of the region to be amplified
extension
the DNA is kept at 72C for 1 minute while the Taq polymerase extends the DNA, adding nucleotides according to the base-pairing rule, until two complete strands are formed
process
the cycle repeats about 30 times, doubling the DNA each time
taq polymerase
is found naturally in Thermus aquaticus which lives in thermal springs and hence doesn’t denature at high temps
nucleotides
also may be called dNTP’s
PCR components
- DNA sample
- primers
- nucleotides
- taq polymerase
- mix buffer
- PCR tube
the number of DNA strands double after each cycle
1 cycle- 2 strands
2 cycle- 4 strands
3 cycles- 8 strands
gel electrophoresis
a technique which sorts DNA fragments by length
- analyzing technique
when is gel electrophoresis used?
it is often used after PCR to check that the process worked and that the correct gene was amplified
gel electrophoresis process
- a gel is made with small pores - allowing DNA fragments to move through
- DNA samples are placed into small wells in the gel
- since DNA is negatively charged (due to it containing phosphate), it moves towards the positive electrode
- smaller fragments move faster and travel further, while larger fragments move more slowly
- a dye makes the DNA bands visible under UV light
a marker / ladder
often run in one or more lanes of gel
- contain DNA fragments of known size
- allow the size of other fragments to be determined
- allow the scientist to determine if the gel ran correctly
gel electrophoresis in DNA profiling
DNA profiling-
a technique that uses variable (non- coding) regions of DNA to identify people.
when is DNA profiling useful
- to identify who has been at a crime scene
- to identify people killed by natural disasters
- to determine who the biological parent of a child are
