Module 6: Section 4 - Manipulating Genomes Flashcards
What is a polymerase chain reaction? (PCR)
the PCR can be used to select a fragment of DNA and amplify it to produce millions of copies in just a few hours
Explain the seven steps of PCR
1) a reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase
2) the DNA mixture is heated to 95 degrees C to break the hydrogen bonds between the two strands of DNA. DNA polymerase doesn’t denature even at this high temperature - this is important as it means many cycles of PCR can be carried out without having to use new enzymes each time
3) the mixture is then cooled to between 50 and 65 degrees so the primers can bind (anneal) to the strands
4) the reaction mixture is heated to 72 degrees C, so DNA polymerase can work
5) the DNA polymerase lines up free DNA nucleotides alongside each template strand. Complementary base pairing means new complementary strands are formed
6) two new copies of the fragment of DNA are formed and one cycle of PCR is complete
7) the cycle starts again, with the mixture being heated with 95 degrees C and this time all four strands are used as templates
What is electrophoresis?
electrophoresis is a procedure that uses an electrical current to separate out DNA fragments, RNA fragments, or proteins depending on their size
Restriction enzymes can be used to cut out DNA fragments. Please explain in six steps how this occurs
1) some sections of DNA have palindromic sequences of nucleotides. These sequences consist of antiparallel base pairs
2) restriction enzymes are enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places
3) different restriction enzymes cut at different specific recognition sequences, because the shape of the recognition sequence is complementary to an enzyme’s active site
4) if recognition sequences are present at either side of the DNA fragment you want, you can use restriction enzymes to separate it from the rest of the DNA
5) the DNA sample is incubated with the specific restriction enzyme, which cuts the DNA fragment out via a hydrolysis reaction
6) sometimes the cut leaves sticky ends - small tails of unpaired bases at each end of the fragment. Sticky ends can be used to bind (anneal) the DNA fragment to another piece of DNA that has sticky ends with complementary sequences
How is electrophoresis used to produce DNA profiles?
1) some of an organism’s genome consists of repetitive, non-coding base sequences - sequences that don’t code for proteins and repeat over and over
2) the number of times these non-coding sequences are repeated
