Gene Technology 5.4 Flashcards
How can you use reverse transcriptase to isolate a certain gene?
The enzyme reverse transcriptase makes DNA from an RNA template. The DNA produced is called complementary DNA (cDNA). So mRNA is isolated from cells, then it’s mixed with free DNA nucleotides and reverse transcriptase. This synthesises cDNA.
How can you use restriction endonuclease to isolate a specific sequence?
Restriction endonucleases are enzymes that recognise specific palindromic sequences and cut the DNA at these places. If the recognition sequences are present either side of the DNA fragment you want, you can use Restriction Endonuclease. The DNA sample is incubated with the enzyme which cuts the DNA via a hydrolysis reaction. Sometimes the cut leaves sticky ends.
How do you use PCR to replicate a certain gene?
A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase. The DNA mixture is heated to 95 degrees to break hydrogen bonds between the strands. The mixture is then cooled to 50-65 degrees so the primers can anneal. The reaction mixture is then heated to 72 degrees so DNA polymerase can work. DNA polymerase lines up free DNA nucleotides alongside each template strand. Two new copies of the fragment of DNA are formed and one cycle of PCR is complete.
What are primers?
Short pieces of DNA that are complementary to the bases at the start of the fragment you want
