The PCR The Polymerase Chain Reaction

The PCR is used to make copies of DNA (amplification). Whole genome OR DNA fragments

The Polymerase Chain Reaction Uses a thermal cycler. Repeated cycles of heating, cooling and reheating. Number of DNA strands doubles with each cycle.

The Polymerase Chain Reaction Ingredients: -DNA to be copied -Primers -DNA polymerase -Nucleotides

Primers – bind to DNA to allow DNA polymerase to work DNA Short (18-24 bases) Single stranded Base sequence complementary to each end of the target DNA One sequence for each DNA strand

DNA Polymerase Needed to join sugar-phosphate backbone of new DNA strands. DNA Taq polymerase is derived from bacteria that live in hot springs. Not denatured by high temperatures required for the PCR to work.

The PCR – How it works 1.Heat to 95°C to break H-bonds between nuclotides and separate DNA strands. 2.Cool to around 60°C to allow primers to anneal (bind) to DNA. 3.Heat to 72°C to give DNA Taq polymerase the optimum temperature for it to join the sugar phosphate backbone of each new strand. These stages are repeated times. How many DNA strands will be present after 5, 10 and 20 cycles?

PCR Puzzle Class instructions

Start of lesson Have the following at front of the class: Template

Start of lesson continued Primers Bases

Correct base pairing is critical! Green (Guanine) pairs with yellow (Cytosine) Blue (Adenine) pairs with orange (Thymine)

The devil is in the detail! The 5’ prime and 3’ prime ends of the bases must be round the right way!

Cycle 1 - denature Spilt class in 4 pairs or groups of students Each group will copy one STRAND from the template Thus, it is important to think of a template as two strands rather than one double stranded piece of DNA

Cycle 1 - denature Two groups will do the first cycle Give Group 1 one template strand Give Group 2 the other template strand

Students comes to front to get primer One student from each group comes to front of class to get primer

Students comes to front to get primer If they have double orange in their template, pick one double blue primer The other group takes one green-blue primer

Cycle 1 - Anneal primer Each group adds primer to the template Make sure 5’ to 3’ prime is correct!

Cycle 1 - extend Students take bases required to finish strand And add the bases to complete the copy

Cycle 1 - observations When finished filling in blanks, each group brings product to front of class and put on desk

Cycle 1 - observations Make observations: – How many copies do you get? – How many are just the target sequence (i.e. begin and end with primers)?

Cycle 2 - denature Groups 1 to 4 will do Cycle 2 Give each group a strand:

Cycle 2 – Anneal primer Each group adds appropriate primer to the template strand Make sure 5’ to 3’ prime correct way round!

Cycle 2 - extend Students take it in turns to come to front to take matching base to add to template

Cycle 2 - observations When finished filling in blanks, each group brings product to front of class and put on desk

Cycle 2 - observations Make observations: – How many copies do you get? – How many are just the target sequence (i.e. begin and end with primers)?

Cycle 3 - denature Groups 1 to 4 will do Cycle 3 Give each group two strands:

Cycle 3 – Anneal primer Each group adds appropriate primer to the template strand Make sure 5’ to 3’ prime correct way round!

Cycle 3 - extend Students take it in turns to come to front to take matching base to add to template

Cycle 3 - observations When finished filling in blanks, each group brings product to front of class and put on desk

Cycle 3 - observations How many copies do you get? How many are just the target sequence (i..e begin and end with primers)? What if anything, has happened to the template? How many bases with red, black and white do you now have? How would you calculate the number of copies produced after each cycle?