ch14: biotechnology Flashcards
PCR (polymerase chain reaction)
to take a sequence of DNA and make many copies (doubles the amount of DNA every cycle)
the three steps of PCR
1) denaturation step: incubate target DNA at a HIGH temp to separate the strands (around 95 degrees C); high temp breaks hydrogen bonds; add primers, nucleotides and DNA polymerase
2) annealing step: primers attach to single-stranded DNA during incubation at LOW temp
3) extension step: incubate at INTERMEDIATE temp; DNA polymerase copies the target DNA
restriction endonucleases (restriction enzyme)
enzymes that cleave DNA at specific sites
cut sites are always
palindromes (the letters going 5’-3’ on the leading strand are the same as the letters going 3’-5’)
blunt ends
cut in the middle of a palindrome; results in no overhang; any two DNA molecules cut with blunt end restriction enzymes can be joined together
sticky ends
staggered cut; results in overhang; any two DNA molecules cut with the same restriction enzyme can be joined together (very specific)
DNA is more —— due to the phosphates, so during a gel electrophoresis, the DNA will move towards the —— side
negative; positive
transformation
introduction of DNA from an outside source into a cell
transgenic organisms
organism that is partly or entirely made from transformed cells
clone
genetically identical copy
molecular cloning
isolation of a specific DNA sequence (usually protein coding)
the most flexible and common host for cloning is
e. coli
vector
carries DNA in host and can replicate in the host
plasmids
small, circular chromosomes used for cloning small pieces of DNA
plasmids need three things:
1) origin of replication: allows independent replication
2) selectable marker: allows presence of plasmid to be easily identified
3) multiple cloning site: unique restriction enzyme sites to easily clone in genes
