Polymerase Chain Reaction

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.

The Reaction Mixture

Water and pH buffer DNA to be multiplied RNA Primers Nucleotides ATG C DNA Polymerase (Taq)
A T G

A
T

C C

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

Repeating the cycle

In the next cycle the heating and cooling steps are repeated. The original (red/purple) strands reproduce as per the first cycle. The new strands only duplicate between the primer sites to produce blocks of a set length.
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
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

Continuing the Cycle

The cycle is then repeated over and over again. With each cycle the number of short fragments rapidly increases while the number of larger fragments increases slowly.
Cycle No Long fragments Short Fragments 0 2 0 1 4 0 2 6 2 3 8 8 4 5 6 14 7 16 8 18 9 20 10 22 2026

10 12

22 52 114

240 494 1004

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.