Polymerase Chain Reaction

1. 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.

2. Uses of PCR • DNA sequencing. • Gene cloning. • DNA profiling. • Transformation. • Making artificial genes.

3. 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 areadded.

4. The Reaction Mixture A G A C T C T • Water and pH buffer • DNA to be multiplied • RNA Primers • Nucleotides • DNA Polymerase (Taq) A T G C

5. 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.

6. Splitting DNA • DNA is heated to 950C which causes double stranded DNA to become single stranded.

7. Adding Primers 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’ G C 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’ • 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 600Cto allow the primers to attach (anneal).

8. Adding Nucleotides 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’ A T C C G T A T C G G A A T A G 5’ G C A U A G C C T T A T C C G T A T T C G 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’ • The reaction temperature is raised to 720Cto allow nucleotides to be added. • Polymerase enzymes (Taq) catalyse the addition of nucleotides. • Nucleotides are added in a 5’ to 3’ direction.

9. Repeating the cycle G G C C A A U U A A 5’ 5’ A T C C G T A T C G G A A T A G C G T A T C G G A A T A G G C C T T A T C C G T A T 5’ 5’ G G C C A A U U A A G C C T T A T C C G T A T T C G A • 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.

10. 1 2 3

11. 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.

12. 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.

13. Summary Heat to 950C Heat to 950C Denature DNA Denature DNA Add Nucleotides Add Nucleotides PCR Cycle Cool to 600C Cool to 600C Heat to 720C Heat to 720C Anneal Primers Anneal Primers

14. 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.

15. 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

16. 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.