Genetic Technologies

1. Genetic Technologies http://www.stats.gla.ac.uk/~paulj/tech_genetics.ppt

2. Overview • Why learn about genetic technologies? • The molecular geneticist’s toolkit • Genetic markers • Microarray assays • Telomeres • RNA interference (RNAi)

3. Why learn about genetic technologies?

4. Why learn about genetic technologies? We need to understand the processes that generated the data • Understanding of biology obviously necessary • Understanding of lab techniques will enhance our ability to assess data reliability • Errors in any measurement can lead to loss of power or bias • Some genetic analyses are particularly sensitive to error because • they depend on the level of identity between DNA sequences shared by relatives • the more data is collected, the greater the chance of false differences

5. Why learn about genetic technologies? • What is the probability that the observed genotype is wrong? • Is this probability the same for all observed genotypes? • What impact will a realistic range of errors have on power? Individual Genotype A 177, 179 B 179, 179

6. The molecular geneticist’s toolkit

7. Most genetic technologies are based on four properties of DNA • DNA can be cut at specific sites (motifs) by restriction enzymes • Different lengths of DNA can be size-separated by gel electrophoresis • A single strand of DNA will stick to its complement (hybridisation) • DNA can copied by a polymeraseenzyme • DNA sequencing • Polymerase chain reaction (PCR)

8. Sau3AI GATC CTAG DNA can be cut at specific sites (motifs)by an enzyme • Restrictionenzymes cut double-stranded DNA at specific sequences (motifs) • E.g. the enzyme Sau3AI cuts at the sequence GATC • Most recognition sites are palindromes: e.g. the reverse complement of GATC is GATC • Restriction enzymes evolved as defence against foreign DNA

9. DNA can be cut at specific sites (motifs)by an enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

10. Sau3AI GATC CTAG DNA can be cut at specific sites (motifs)by an enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

11. Sau3AI GATC CTAG DNA can be cut at specific sites (motifs)by an enzyme ACTGTCGATGTCGTCGTCGTAGCTGCT GATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAG CATCGATCGA

12. DNA can be cut at specific sites (motifs)by an enzyme ACTGTCGATGTCGTCGTCGTAGCTGCT TGACAGCTACAGCAGCAGCATCGACGACTAG GATCGTAGCTAGCT CATCGATCGA ACTGTCGATGTCGTCGTCGTAGCTGCTGA TGACAGCTACAGCAGCAGCATCGACGACT TCGTAGCTAGCT AGCATCGATCGA

13. Different lengths of DNA can be separated by gel electrophoresis • DNA is negatively charged and will move through a gel matrix towards a positive electrode • Shorter lengths move faster

14. Different lengths of DNA can be separated by gel electrophoresis • DNA is negatively charged and will move through a gel matrix towards a positive electrode • Shorter lengths move faster

15. S M F Different lengths of DNA can be separated by gel electrophoresis Slow: 41 bp ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA Medium: 27 bp ACTGTCGATGTCGTCGTCGTAGCTGCT TGACAGCTACAGCAGCAGCATCGACGACTAG Fast: 10 bp GATCGTAGCTAGCT CATCGATCGA

16. DD HH HD S M F Different lengths of DNA can be separated by gel electrophoresis Recessive disease allele D is cut by Sma3AI: ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA Healthy H allele is not cut: ACTGTCGATGTCGTCGTCGTAGCTGCTGAGCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTCGCATCGATCGA

17. DD HH HD S M F Different lengths of DNA can be separated by gel electrophoresis

18. A single strand of DNA will stick to its complement ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

19. A single strand of DNA will stick to its complement 60°C ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

20. A single strand of DNA will stick to its complement 95°C ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

21. A single strand of DNA will stick to its complement 60°C ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

22. A single strand of DNA will stick to its complement Fragment frequency Flourescence Fragment length in bp

23. A single strand of DNA will stick to its complement

24. A single strand of DNA will stick to its complement Southern blotting (named after Ed Southern)

25. A single strand of DNA will stick to its complement Southern blotting (named after Ed Southern)

26. A single strand of DNA will stick to its complement

27. A single strand of DNA will stick to its complement

28. A single strand of DNA will stick to its complement

29. DNA can copied by a polymerase enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA

30. DNA polymerase DNA can copied by a polymerase enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA A G T G C A A G C T G G A A G A G T T C T C C C A G T A A G

31. DNA polymerase DNA can copied by a polymerase enzyme ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT TGACAGCTACAGCAGCAGCATCGACGACTAGCATCGATCGA A G T G C A A G C T G G A A G A G T T C T C C C A G T A A G

32. DNA can copied by a polymerase enzyme ACTGTCGATGTCGT

33. DNA can copied by a polymerase enzyme ACTGT ACTGTCGAT ACTGTCGATGT ACTGTCGATGTCGT ACTGTCGATGTCGTCGT ACTGTCGATGTCGTCGTCGT ACTGTCGATGTCGTCGTCGTAGCT ACTGTCGATGTCGTCGTCGTAGCTGCT ACTGTCGATGTCGTCGTCGTAGCTGCTGAT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCT ACTGTCGATGTCGTCGTCGTAGCTGCTGATCGTAGCTAGCT

34. DNA can copied by a polymerase enzyme ACTGTCGATGT ACTGTCGATG ACTGTCGAT ACTGTCGA ACTGTCG ACTGTC ACTGT T G T A G C T Time Fluorescence T C G A T G T etc Fluorescence Time

35. DNA can copied by a polymerase enzyme

36. DNA can copied by a polymerase enzyme

37. DNA can copied by a polymerase enzyme

38. DNA can copied by a polymerase enzyme Polymerase chain reaction (PCR) • A method for producing large (and therefore analysable) quantities of a specific region of DNA from tiny quantities • PCR works by doubling the quantity of the target sequence through repeated cycles of separation and synthesis of DNA strands

39. DNA can copied by a polymerase enzyme

40. Heat resistant DNA polymerase Forward primer Reverse primer G, A, C, T bases DNA template A thermal cycler (PCR machine) DNA can copied by a polymerase enzyme G A C T

41. DNA can copied by a polymerase enzyme

42. DNA can copied by a polymerase enzyme

43. DNA can copied by a polymerase enzyme

44. DNA can copied by a polymerase enzyme In the words of its inventor, Kary Mullis… • PCR can generate 100 billion copies from a single DNA molecule in an afternoon • PCR is easy to execute • The DNA sample can be pure, or it can be a minute part of an extremely complex mixture of biological materials • The DNA may come from • a hospital tissue specimen • a single human hair • a drop of dried blood at the scene of a crime • the tissues of a mummified brain • a 40,000-year-old wooly mammoth frozen in a glacier.

45. DNA can copied by a polymerase enzyme

46. The molecular geneticist’s toolkit • Specific DNA-cutting restriction enzymes • DNA size separation by gel electrophoresis • Hybridisation using labelled DNA probes • Synthesis of DNA using DNA polymerase (PCR)

47. Genetic markers

48. Genetic markers • What are they? • Variable sites in the genome • What are their uses? • Finding disease genes • Testing/estimating relationships • Studying population differences

49. Eye colour

50. ABO blood group