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A process used to artificially multiply a chosen piece of genetic material. May also be known as DNA amplification. One strand of DNA may yield 230 strands or more.
Uses of PCR
DNA sequencing. Gene cloning.
DNA profiling.
Transformation. Making artificial genes.
DNA Selection
DNA is selected either as a complete chromosome or a fragment. Primers are constructed that will bind within a desired region (purple). Additional reaction chemicals are added.
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PCR Machine
When mixed, the reaction tubes are placed into a PCR machine. The machine can be set to accurately control reaction times and changes in temperature.
Splitting DNA
DNA is heated to 950C which causes double stranded DNA to become single stranded.
Adding Primers
RNA primers are prepared that base pair with a selected sequence of DNA. Two primers must be used.One for each strand of the DNA. The reaction temperature is lowered to 600C to allow the primers to attach (anneal).
5 T A G G C A T A G C C T T A T C C G T A T T C G T G C A U A 5 5 GC A U A A T C C G T A T C G G A A T A G G C A T A A G C A 5
Adding Nucleotides
The reaction temperature is raised to 720C to allow nucleotides to be added. Polymerase enzymes (Taq) catalyse the addition of nucleotides. Nucleotides are added in a 5 to 3 direction.
5 T A G G C A T A G C C T T A T C C G T A T T C G T
A T C C G T A T C G G A A T A GG C A U A 5
GC A U A GC C T T A T C C GT A T T C GA A T C C G T A T C G G A A T A G G C A T A A G C A 5
GC A U A GC C T T A T C C GT A T
C G T A T C G G A A T A GG C A U A 5 GC A U A GC C T T A T C C GT A T T C GA
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22 52 114
30 Cycles
After 30 cycles, if replication has occurred fully, a total of 2,147,483,648 strands could be produced. All but 62 will be the short length of the desired DNA fragment.
Summary
Heat to 950C Add Nucleotides Denature DNA
PCR Cycle
Heat to 720C Anneal Primers Cool to 600C
Questions
What does PCR stand for? Polymerase chain reaction. The PCR reaction may also be known as? DNA amplification. In addition to DNA what are the key reactants needed for PCR? pH Buffer, Nucleotides, Taq enzyme, Primers. The first step in the PCR reaction is to heat to 900C. What does this do? Splits double stranded DNA into single strands.
Questions Continued
The next step is to lower the temperature to 600C. What is the purpose of this? Allows primers to be added. What name is given to the process of joining primers to DNA? Annealing. At what temperature are nucleotides added? 720C What name is given to the polymerase enzyme used? Taq
Questions continued
In which direction are nucleotides added? 5 to 3 Why is the cycle repeated many times? To allow the rapid build up of fragment numbers. Name five key uses of PCR. DNA sequencing, gene cloning, DNA profiling transformation, making artificial genes.