8B Amplifying DNA Fragments Flashcards
Step 1 of in vitro cloning
The DNA is inserted into a vector
The vector DNA is cut open using the same restriction nuclease enzyme that was used to isolate the DNA containing the target gene so the sticky ends and complementary to the ends of the DNA fragment containing the gene
The vector DNA and the DNA fragment are mixed together with DNA ligase that joins the sticky ends together by a process called litigation
The new combination of bases
Define vector
An organism used to transfer DNA into a host cell
Function of DNA ligase
Joins the sticky ends of the DNA fragment to the vector DNA
Step 2 of in vitro cloning
The vector with the recombinant DNA is used to transfer the gene into host cells
If a plasmid vector has be used a host cell has to be persuaded to take up the vector and its DNA
With a bacrophage the bacrophage will infect the host cell by infecting the host cell with its DNA the phage DNA then integrates into the bacterial DNA
Define transformed cell
Host cells that take up the vectors containing the gene of interest
Marker gene function
to identify transformed cells
What do any transformed host cells contain
the marker gene and the gene to be cloned
What can marker genes code for
antibiotic resistance or UV fluorescence
Define promotor regions
DNA sequences that tell RNA polymerase when to start without this and terminator regions the right protein won’t be transcribed
Define terminator regions
DNA sequences that tell RNA polymerase when to stop
What does a pcr reaction mixture contain
The DNA sample, free nucleotides, primers and DNA polymerase
Define primers
short pieces of DNA that are complementary to the bases at the start of the fragment
What temperature is the DNA mixture heated to the first time
75 degrees Celsius
What temperature is the mixture cooled to
between 50 and 65 degrees Celsius so the primers can bind to the strand
What temperature is the reaction mixture heated to so that DNA polymerase can work
72 degrees Celsius
What does DNA polymerase do in the reaction mixture
It lines up free nucleotides alongside each template strand and joins the nucleotides together by specific base pairing means new complementary strands are formed two new strands are formed and one cycle is complete
What temperature is the reaction mixture heated to after the first cycle
95 degrees Celsius
What happens to the amount of DNA after each PCR cycle
it doubles
