The PCR The Polymerase Chain Reaction The Polymerase

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The PCR The Polymerase Chain Reaction

The PCR The Polymerase Chain Reaction

The Polymerase Chain Reaction • The PCR is used to make copies of DNA

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,

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

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

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

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

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 20 -35 times. How many DNA strands will be present after 5, 10 and 20 cycles?

PCR Puzzle Class instructions

PCR Puzzle Class instructions

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

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

Start of lesson continued • Primers • Bases

Start of lesson continued • Primers • Bases

Correct base pairing is critical! • Green (Guanine) pairs with yellow (Cytosine) • Blue

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

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

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

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

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

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 •

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

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

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?

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 •

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

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

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

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?

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 •

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

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

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

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

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?

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