Manipulating genomes. Flashcards
What are restriction enzymes?
Endonuclease enzymes that cleave DNA molecules at specific recognition sites
What does PCR stand for?
Polymerase chain reaction
What does PCR do?
‘amplifies’ - it increases the quantity of DNA.
List what is included in the mixture in PCR
- DNA sample
- Free nucleotides
- Primers
- DNA polymerase
What are primers?
Short pieces of DNA that are COMPLEMENTARY to the bases at the start of the fragment wanted.
Outline the process of the PCR (polymerase chain reaction)
- (1) A reaction mixture is set up that contains: the DNA sample, free nucleotides, primers and DNA polymerase enzymes.
- (2) The DNA mixture is heated to 95 degrees in order to break the hydrogen bonds between the 2 strands of DNA.
- (3) The mixture is cooled to 55 degrees so primers can ANNEAL (bind) to the strands.
- (4) The reaction mixture is reheated again to 72 degrees so DNA polymerase can work at its optimum
- (5) The DNA polymerase lines up free nucleotides alongside each template strand –> complementary base pairing means new complementary strands are formed.
- (6) Two new copies of DNA are formed and the first PCR cycle is complete.
- (7) The cycle repeats, being reheated to 90 - all 4 strands (the two original and two new) are used as templates.
Why does the fact that DNA polymerase not denaturing at a high temperature prove beneficial in PCT?
As DNA polymerase DOES NOT denature, even at high temperatures of 95 degrees, it proves important as it allows many cycles of PCR to occur without needing new enzymes.
What are restriction enzymes also referred to as?
Restriction endonucleases
What do restriction enzymes allow?
They enable DNA fragments / genes to be extracted from an organisms DNA.
What is meant by a ‘palindromic sequence’ ?
DNA that has the same order of bases when read backwards on the opposite strand.
