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level: 21.3 In vitro gene cloning - the polymerase chain reaction

Questions and Answers List

level questions: 21.3 In vitro gene cloning - the polymerase chain reaction

QuestionAnswer
Polymerase chain reaction- Invitro - DNA amplification - Application = amplifying DNA of crime scene - It allows thousands of copies of DNA to be cloned in a short period of time
DNA polymerase (taq polymerase)- The enzyme that joins nucleotides to form double stranded DNA - Converts single-stranded DNA into double-stranded
Primer- A short piece of complementary DNA that is required for DNA polymerase to attach
Thermocycler- A machine which automates the process by heating then cycling through different temperatures (95, 55, 72)
Why not use human DNA polymerase?- Denatures - Breaks bonds in 3o structure, change its shape, no longer complementary, can't bind
How would the 3o structure of this enzyme be different to human DNA polymerase?- More disulphide bridges in 3o structures therefore less likely to break
Why is the DNA heated to 95oC?- Breaks H bonds, separate the strands - To allow primers to attach
Why are primers required?- To allow DNA to be double stranded so DNA polymerase can attach - Identifies specific base sequence we can copy - (Enzyme) needs starting strand onto which to attach nucleotides
Why is there excess primer added?- Stop original DNA strands from reattaching
equation2^n n = number of cycles
Why 2 different primers?- DNA mol. anti parallel so different base sequences - So primers will be complementary to the specific base sequence on either strand
Process- Denaturation – DNA is heated to 95°C which breaks the h bonds, strands seperate - Annealing - the temperature is decreased to 52°C so that primers can join to their complementary bases at the end of the DNA fragment - Elongation / Extension – the temperature is increased to 72°C, as this is the optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double-stranded DNA molecules