DNA manipulation techniques and applications #4 Flashcards
in natural circumstances DNA polymerase is used to
make new copies of DNA during cell division
when DNA is condensed
not much synthesis occurs
endonucleases
enables the bacteria to cut DNA of invading viruses
in genetic engineering DNA polymerase is used to
make copies of DNA through a process called PCR
they can be cut in 2 ways
- sticky end
- blunt end
restriction enzymes (endonucleuses)
cuts DNA at specific base sequence to produce smaller pieces of DNA called restriction fragments
HaeII
a restriction enzyme which cleaves (cuts the sequence GGCC.
- it cuts through the DNA and forms blunt ends
blunt ends
can join to other segments of DNA but it is random rather than specific as these fragments can connect to any other fragment of DNA;
EcoR1
recognizes the sequence GAATTC but does not cut through the DNA completely and leaves STICKY ENDS
-these DNA pieces will bind to other pieces of DNA which have been cut with the same restriction enzyme, not necessarily of the same species
ligases
enzymes used to join 2 different pieces of DNA
- DNA sticky tape
phosphodiester bond
holds the sides of the phosphates and sugar together
- created by DNA ligase
hydrogen bonds
holds complementary bases together in a pair
ligases .2
they form a phosphodiester bond between the 3’ end of a nucleotide on one fragment with the 5’ end of a nucleotide on another fragment
ligase to join DNA
- more stable
hydrogen bonds will form between the exposed nucleotides and ligase will form bonds between the nucleotides of the two fragments
if you cut a plasmid in one place..
you get one fragment of DNA
if you cut linear DNA in one place
you get two fragments
