Key area 2: Replication of DNA

1. Key area 2: Replication of DNA

2. How is DNA replicated? Now you know the structure of DNA... …how is it copied?

3. Why do cells need to copy their DNA? DNA is copied during cell division (mitosis) to ensure that new cells have the same number of chromosomes and to ensure that all cells have the same genes.

4. Hypothesise… Take a 5 minutes to discuss in groups ways in which DNA might copy itself. Use colouring pencils to put your ideas on the idea sheet. Key Original DNA New Copied DNA

5. Three possible hypotheses

6. Meselson and Stahl Meselson and Stahl were two scientists who, in 1958, carried out an ingenious experiment to solve this question.

7. This is their experiment: They grew E. coli bacteria in “Heavy” nitrogen. This nitrogen was used to make DNA. They grew the cells first in “heavy nitrogen” then switched them to “light nitrogen” .

8. When you spin DNA with “Heavy nitrogen” in a ultracentrifuge it sinks to the bottom. “Light nitrogen” stays at the top. DNA with LIGHT nitrogen only DNA with both heavy AND light nitrogen DNA with HEAVY nitrogen only In groups fill in a results prediction sheet. Each group will be allocated either: Conservative, Semi-conservative or Dispersive.

9. The results… So which model was correct?

10. Summary In your jotter, create a summary of: The 3 models of DNA replication Medelson & Stahl’s experiment design Their results.

12. DNA is a unique molecule because it can direct its own replication and reproduce itself. DNA replicates by semi-conservative replication.

13. A C T G C A A C T G C A T G A C G T T G A C G T DNA parental strand composed of two complementary strands STEP 1: Hydrogen bonds between the bases break – separating the strands

14. A C T G C A T G A C G T T T C C C C A A G G T T A A G G STEP 2: Free nucleotides start to line up with complementary nucleotides

15. A C T G C A A C T G C A T G A C G T T G A C G T STEP 3: Sugar-phosphate bonds form. Two DNA molecules identical to the parental molecule have been formed.

16. Enzyme control of DNA replication DNA replication is a complex process involving many enzymes. The enzyme DNA polymerase controls the formation of the sugar-phosphate bonds when making the new strand.

17. The leading strand DNA polymerase can only join nucleotides onto the 3’ end of a growing DNA strand. Therefore…

18. DNA polymerase enzyme Direction of replication Start of complementary strand of replicated DNA 5’ end of DNA strand Leading strand of replicated DNA Replication of the leading strand of DNA 3’ end of DNA strand Primer

19. After the hydrogen bonds break, the DNA unzips. A DNA primer (a short stretch of complementary DNA) attaches to the start of the piece of DNA being copied. DNA polymerase the attaches free nucleotides to the 3’ end of the primer. This continuous process till leading strand is copied.

20. 3’ end of DNA strand Replication of the lagging strand of DNA Primer DNA polymerase Ligase 5’ end of DNA strand

21. DNA polymerase can only add onto the 3’ end of a primer. So for the other strand: Many primers attach along the strand. These are extended by the DNA polymerase. The fragments are then joined by the enzyme ligase This is a discontinuous process creating the lagging strand.

22. Replication bubbles and forks

23. When copying a long chromosome many replication forks operate simultaneously to speed up the replication process.

24. Requirements for DNA replication For DNA replication to occur, the nucleus must contain: • DNA (to act as the template) • Primers • A supply of the 4 types of nucleotide • DNA polymerase and ligase enzymes • A supply of ATP (energy)

25. The polymerase chain reaction (PCR)

26. Prior knowledge • The structure of DNA. • DNA replication process.

27. What is PCR? PCR (Polymerase chain reaction) was developed by Kary Mullis in the mid-1980s. For which he received the Nobel Prize. It has revolutionized molecular biology.

28. What is PCR? PCR allows specific sections of DNA to be amplified in vitro (replicated out with a cell in a test tube (in vitro = in glass)).

29. Millions of copies of a specific piece of DNA can be created in a few hours in a thermocycler.

30. The first cycle 5’ 3’ Single copy of DNA 3’ 5’ Step 1: The DNA is heated at approx. 95 oC for a few seconds. This causes the DNA to denature and the strands to separate.

31. 5’ 3’ 5’ 3’ PCR primer 3’ 5’ 3’ 5’ Step 2: The DNA is cooled to approx. 50-65 oC for a few seconds. This makes short primers to bond to the separated DNA strands.

32. 5’ 3’ 3’ 5’ 3’ 5’ 3’ Step 3: The DNA is heated again to approx. 72oC for a few minutes. This allows a heat-tolerant DNA polymerase to replicate the DNA. 5’

33. 5’ 3’ 3’ 5’ 3’ 5’ 3’ Step 4: Heat the DNA up to 95 oC again. 5’

34. 5’ 3’ 3’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 3’ Step 5: Cool to between 50 – 65 oC again. The primers now bond to the original fragments and the copies. 5’

35. 5’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 3’ 5’ Step 6: Heat to 72 oC again. The DNA polymerase copies the DNA again. The process is copied over and over again for roughly 20-30 cycles.

37. Requirements for PCR Sequence specific primers – these are designed by the scientist and can be manufactured by a machine. The sequence for primers can be designed by looking at the published genome sequences.

38. Primers • Supply of nucleotides • pH buffer • Mg2+ - DNA polymerase co-factor (makes the polymerase work better)

39. Uses of PCR • DNA Profiling PCR helps to rapidly identify people. Specific areas of DNA known to vary between individuals is amplified. Giving different sized fragments in different people.

40. 2. Disease detection DNA sequences that are known to indicate certain genetic disorders or diseases are amplified using PCR for the purposes of diagnosis.

41. 3. Archeological analysis Ancient DNA, degraded over the years, can be amplified and used in archaeological, paleontological and evolutionary research.

42. 5. Population studiesAnalysis of human or other species’ population genetics can be rapidly performed using PCR analysis. 6. Sequencing DNA sequences can be worked out.

43. Key concepts • Small sections of DNA can be replicated in vitro using the PCR. • PCR manipulates the natural process of DNA replication. • PCR is now an automated technique widely used in many areas of research and industry. • PCR requires template DNA, Taq polymerase, di-deoxynucleic acids with each of the four DNA bases, Mg2+, primers and a buffer. • PCR involves continuous and repeated cycles of heating and cooling.