1. Summary of: DNA and its Role in Heredity Mohammed Ghorwal Maria Steingrimsdóttir Claus Jensen Sheida Tehrani Next weeks summary: Alexandre Simoens Lea Jensen Malte Bukrinski Majken Frederiksen

2. Outline of today Short lecture on PCR: –What happens in the PCR tube Copy-and-paste PCR exercise in power point Exercises regarding PCR similar to exam questions Virtual laboratory –solving a murder using PCR

3. PCR A technique for making multiple copies of a specific DNA sequence / amplify a specific DNA sequence Why? For medical purposes: -Does the patient have the disease-causing variation of a gene? -For determining which type of bacteria is involved in an outbreak. -Tissue typing of patient and donor before bone marrow transplantation. Crime lab forensics: -Paternity cases. -Identification of a perpetrator. Research: -Did the cloning succeed? ….AND MUCH MUCH MORE ….

4. Remember the 5’ and 3’ ends

5. T A T C A A A T C A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ A A A T A A T G T T T C G C C T C G 5’ 3’ C A C A G 5’ 3’ C A C A G 5’ 3’ C A C A G 5’3’ C A C A G T G T C C 5’3’ T G T C C 5’ 3’ T G T C C 5’ 3’ T G T C C 5’ 3’ DNA polymerase DNA polymerase A T A G T T T A G T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’

6. T A T C A A A T C A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ A T A G T T T A G T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ 5’ 3’ C A C A G T G T C C 5’ 3’ T G T C C 5’ 3’ 5’ 3’ C A C A G A T A G T T T A G T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T C A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ A A A T A A T G T T T C G C C T C G DNA polymerase T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T C A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’

7. A T A G T T T A G T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T C A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T C A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ 5’ 3’ C A C A G T G T C C 5’ 3’ T A T C A A A T C A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ 5’ 3’ C A C A G T G T C C 5’ 3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 3’ 5’ T A T C A A A T C A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 3’ 5’ 3’ A C A G G T C T A C T T C T A A C A G T C A C A G 5’ T G T C C A G A T G A A G A T T G T C A G T G T C 3’ 5’

8. 3’ C A C A G T G T C C 5’ 3’ A C A G G T C T A C T T C T A A C A G T C A C A G 5’ T G T C C A G A T G A A G A T T G T C A G T G T C 3’ 5’ T G T C C A G A T G A A G A T T G T C A G T G T C 3’ 5’ 3’ A C A G G T C T A C T T C T A A C A G T C A C A G 5’ T A T C A A A T C A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ A T A G T T T A G T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ C A C A G T G T C C

9. Cut and paste PCR During the exercise, you should copy sequences from this slide Original DNA Primers By-product DNA PCR product

10. Which ingredients are necessary for PCR? Step 1 (first cycle): Heating Imagine that all necessary ingredients have been mixed in a PCR tube. Below, the original DNA molecule is shown. It is to function as a template during the PCR. The PCR tube is heated to > 90° C. Show what the heating does to the original DNA molecule. Original DNA Illustration A – the original DNA after heating Heating T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ A T A G T T T A C T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’

11. Step 2 (first cycle): Cool-down and primer binding The mixture is cooled-down, which enables binding of the two primers to the DNA strands in illustration A. Transfer illustration A from the previous slide and a set of primers from the first slide. Then show where the two primers bind on the original DNA strands. Illustration B – the original DNA with bound primers T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ A T A G T T T A C T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ 5’3’ C A C A GT G T C C 5’ 3’

12. Step 3 (first cycle): Synthesis The DNA polymerase synthesizes the two by-product DNA strands starting at the 3’ ends of the primers and using dNTPs. Transfer illustration B from the previous slide and copy a set of the by-product DNA strands from the first slide. Show how each DNA molecule now consists of an original DNA strand and a by-product DNA strand (delete the primers, since they are incorporated into the by-product DNA strands). Illustration C – The by-product DNA strands have been synthesized T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ A T A G T T T A C T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ End of 1 st cycle

13. Step 1 (second cycle): Heating The mixture is again heated to > 90° C. Transfer illustration C from the previous slide and show what happens to the two DNA molecules during the heating. Illustration D – Heating of the DNA molecules that have been generated during the first cycle T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ A T A G T T T A C T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’

14. Step 2 (second cycle): Cool-down and primer binding The mixture is cooled-down, which enables binding of the primers. Transfer illustration D and two sets of primers from the first slide. Then show where the, all in all, four primers will bind. Illustration E – Primer binding T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ A T A G T T T A C T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ 5’3’ C A C A GT G T C C 5’ 3’ 5’3’ C A C A G T G T C C 5’ 3’

15. Step 3 (second cycle): Synthesis Again, the DNA polymerase synthesizes new DNA strands starting at the 3’ ends of the primers. Transfer illustration E from the previous slide and copy a set of by-product DNA strand and a set of PCR product DNA strands from the first slide. Show how in total there is now generated four DNA molecules (two consisting of an original DNA strand and a by-product DNA strand and two consisting of a by-product DNA strand and a PCR DNA strand). Delete the primers, since they are incorporated into the by-product DNA strands and the PCR product DNA strands. Illustration F – Generation of by-product DNA strands and PCR product DNA strands. T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ A T A G T T T A C T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ A C A G G T C T A C T T C T A A C A G T C A C A G 5’ T G T C C A G A T G A A G A T T G T C A G T G T C

16. Step 1 (third cycle): Heating The mixture is re-heated to > 90° C. Transfer illustration F and show what happens to the DNA molecules. Illustration G – Heating T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ A T A G T T T A C T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T C A G A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T C A G A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ A C A G G T C T A C T T C T A A C A G T C A C A G 5’ T G T C C A G A T G A A G A T T G T C A G T G T C 3’ 5’

17. Step 2 (third cycle): Cool-down and primer binding Transfer illustration G. Copy 4 sets of primers from the first slide. Show where they bind. Illustration H – Primer binding T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ A T A G T T T A C T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ A C A G G T C T A C T T C T A A C A G T C A C A G 5’ T G T C C A G A T G A A G A T T G T C A G T G T C 3’ 5’ 3’ C A C A GT G T C C 5’ 3’ 5’3’ C A C A GT G T C C 5’ 3’ 5’3’ C A C A GT G T C C 5’ 3’ 5’3’ C A C A GT G T C C 5’ 3’

18. Step 3 (third cycle): Synthesis Transfer iIllustration H. Copy a set of by-product DNA strands and 3 sets of PCR product DNA strands. Show how in total there is now generated 8 DNA molecules (each consisting of two strands). Illustration I – in total, 8 DNA molecules have been generated T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G T C G G G C G T T T 5’3’ A T A G T T T A C T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’ A C A G G T C T A C T T C T A A C A G T C A C A G 5’ T G T C C A G A T G A A G A T T G T C A G T G T C 3’ 5’ 3’ C A C A G 3’ A C A G G T C T A C T T C T A A C A G T C A C A G 5’ T G T C C A G A T G A A G A T T G T C A G T G T C 3’ 5’ 3’ A C A G G T C T A C T T C T A A C A G T C A C A G 5’ T G T C C A G A T G A A G A T T G T C A G T G T C 3’ 5’ 3’ A C A G G T C T A C T T C T A A C A G T C A C A G T G T C C A G A T G A A G A T T G T C A G T G T C 3’ 5’ T A T C A A A T G A C A G G T C T A C T T C T A A C A G T C A C A G 5’3’ T G T C C A G A T G A A G A T T G T C A G T G T C A G C C C G C A A A 5’ 3’

19. That was the end of the third cycle. It is now impractical to mimic more cycles. Fill out the below table with regards to how many of the different DNA molecules that are present at the end of each of the cycles. The end of cycle no. (n) Number of DNA molecules consisting of an original DNA strand and a by-product DNA strand Number of DNA molecules consisting of a by- product DNA strand and a PCR product DNA strand (2*n – 2) Number of DNA molecules consisting of a double- stranded PCR product. (2^n – 2*n) 1200 2220 3242 4268 52822 621052 7212114 8214240 1002198 1,267*10E30

20. Agarose gel When the PCR has finished, the mixture is run on an agarose gel, which makes it possible to estimate the size of the PCR product. Show where you would expect to see a band after 100 cycles of our PCR. Ladder containing DNA fragments of known length is out into this lane 40 bp 30 bp 20 bp 10 bp After 100 cycles, the mixture is put into this lane