2. 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 2 30 strands or more.

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

4. 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.

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

6. 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.

7. Splitting DNA DNA is heated to 95 0 C which causes double stranded DNA to become single stranded.

8. 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 60 0 C to allow the primers to attach (anneal). G C A U A 5’ G C A U A 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 G G C A T A A G C A

9. Adding Nucleotides The reaction temperature is raised to 72 0 C to allow nucleotides to be added. Polymerase enzymes (Taq) catalyse the addition of nucleotides. Nucleotides are added in a 5’ to 3’ direction. A T C C G T A T C G G A A T A G 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 G C C T T A T C C G T A T T C G A 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

10. 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 G G C A U A 5’ 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 T C G A G C A U A G C A U A G C A U A G C C T T A T C C G T A T

11. 1 3 2

12. 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 No012345678910 Long fragments246810121416182022 Short Fragments 0028225211424049410042026

13. 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.

14. Summary Heat to 95 0 C Denature DNA Cool to 60 0 C Anneal Primers PCR Cycle Add Nucleotides Heat to 72 0 C Heat to 95 0 C Denature DNA Cool to 60 0 C Anneal Primers Heat to 72 0 C Add Nucleotides

15. 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 90 0 C. What does this do? Splits double stranded DNA into single strands.

16. Questions Continued The next step is to lower the temperature to 60 0 C. 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? 72 0 C What name is given to the polymerase enzyme used? Taq

17. 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.