2. 1 Lecture 9 & 10 Dr. Aparna Islam

3. PRINCIPLES OF DNA ISOLATION & PURIFICATION 2

4. Purpose of DNA Extraction To obtain DNA in a relatively purified form which can be used for further investigations, i.e. PCR, sequencing, etc 3 DNA can be isolated from any nucleated cell.

5. Basic Protocol Most DNA extraction protocols consist of two parts 1.A technique to lyse the cells gently and solubilize the DNA 2.Enzymatic or chemical methods to remove contaminating proteins, RNA, or macromolecules In plants, the nucleus is protected within a nuclear membrane which is surrounded by a cell membrane and a cell wall. Four steps are used to remove and purify the DNA from the rest of the cell. 1.Lysis 2.Precipitation 3.Wash 4.Re-suspension 4

6. In forensic Science Sources of DNA include Blood Buccal cells Cultured cells (plant and animal) Bacteria Biopsies Forensic samples i.e. body fluids, hair follicles, bone & teeth roots. 5

7. DNA isolation is a routine procedure to collect DNA for subsequent molecular analysis. There are three basic steps in a DNA extraction: Cell disruption:- This is commonly achieved by grinding or sonicating the sample. Removing membrane lipids by adding a detergent. Isolation of DNA:- Removing proteins by adding a protease (optional but almost always done). Precipitating the DNA :-usually ice-cold ethanol or isopropanol is used. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol soluble salt. 6

8. 7 7 Lysozyme EDTA: removes magnesium ions that is required for integrity of the cell membrane and also chelates MgCl 2 and thereby inhibits Dnase. SDS/CTAB/Lauryl Sarcosinate: remember detergents wash lipid substances! NaOH: causes hydrolysis of the membrane (alkaline lysis) NaCl: causes the cell to rupture Growing and harvesting a bacterial culture

9. 8 8 1:1 Phenol Chloroform mixture: The organic layers captures the protein. The aquous layer has the DNA. DNA is charged negatively, so forms hydrogen bond with water. Also protease can be added to degrade protein contamination. Purification of DNA from cell extract (Get rid of protein!)

10. 9 9 Alcohol addition: Isopropanol or ethanol addition causes the polarity of the solution to change. So DNA precipitates! Precipitate DNA from aqueous layer

11. Break down the cell wall and membranes Centrifuge to separate the solids from the dissolved DNA Precipitate the DNA using isopropanol Centrifuge to separate the DNA from the dissolved salts and sugars Wash the DNA pellet with Ethanol and dry the pellet Dissolve DNA Overview of DNA Extraction 10

12. Sample for DNA extraction Lysis of cells with detergent + enzyme in salt buffer Removal of cellular proteins Precipitation of nucleic acids with ethanol NEXT STEP IS Quantification and purity measurement of DNA Summary 11

13. Checking the Quality of your DNA The product of your DNA extraction will be used in subsequent experiments Poor quality DNA will not perform well in PCR, restriction digestion or any other experiments You will want to assess the quality of your DNA extraction using the following simple protocol: Mix 10 µl of DNA with 10 µl of loading buffer Load this mixture into a 1% agarose gel Analyze results (the following slides provide guidance) 12

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15. What is Gel Electrophoresis? Gel electrophoresis separates molecules on the basis of their charge and size. The charged macromolecules migrate across a span of gel because they are placed in an electrical field. The gel acts as a sieve to retard the passage of molecules according to their size and shape. 14

16. Gel electrophoresis 15 The basic principle is that DNA, RNA, and proteins can all be separated by means of an electric field. In agarose gel electrophoresis, DNA and RNA can be separated on the basis of size by running the DNA/RNA through an agarose gel. Proteins can be separated on the basis of size by using an SDS-PAGE gel, or on the basis of size and their electric charge by using what is known as a 2D gel electrophoresis.

17. http://www.life.uiuc.edu/molbio/geldigest/electro.html#run 16

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20. 1 kbp and 100 bp ladders Genomic DNA of 5 species of cereals Expected Results in a Research Lab Below is an agarose gel that has 5 genomic DNA samples from various plants. Note that the isolated DNA (genomic DNA) runs at a very high molecular weight and as a clear, thick band. This DNA was extracted in a research lab under optimal conditions 19

21. Many uses of restriction enzymes… we can cut DNA with restriction enzymes… – After cutting DNA from different people… or different organisms… we can compare it – why? forensics medical diagnostics paternity evolutionary relationships and more… 20

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23. http://www.life.uiuc.edu/molbio/geldigest/electro.html#run 22

24. 23 DNA is colorless so we need to stain it to visualize

25. Ethidium bromide is fluorescent in UV light 24

26. 25 What do the bands in the drawing of the agarose gel represent? Which band(s) traveled slowest? Which band(s) traveled fastest? On the drawing, label the positive and negative ends of the gel. How many bands are shared in common by all of the individuals? Are there any bands which are unique to only one individual? If so, which one?

27. Restriction Enzymes 26

28. Restriction Endonucleases Also called restriction enzymes 1962: “molecular scissors” discovered in in bacteria E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA 3,000 enzymes have been identified, around 200 have unique properties, many are purified and available commercially 27

29. Restriction Endonucleases Named for bacterial genus, species, strain, and type Example: EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1 28

30. Restriction Endonucleases Recognition sites have symmetry (palindromic) “Able was I, ere, I saw Elba” Bam H1 site: 5’-GGATCC-3’ 3’-CCTAGG-5’ 29

31. Restriction Endonucleases Enzymes recognize specific 4-8 bp sequences Some enzymes cut in a staggered fashion - “sticky ends” EcoRI 5’…GAATTC…3’ 3’…CTTAAG…5’ Some enzymes cut in a direct fashion – “blunt ends” PvuII 5’…CAGCTG…3’ 3’…GTCGAC…5’ 30

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33. Restriction Endonucleases Why don’t bacteria destroy their own DNA with their restriction enzymes? 32

34. Methylation 33

35. Uses for Restriction Enzymes RFLP analysis (Restriction Fragment Length Polymorphism) DNA sequencing DNA storage – libraries Transformation Large scale analysis – gene chips 34

36. 35 Polymerase Chain Reaction (PCR)

37. Polymerase chain reaction (PCR) The polymerase chain reaction is an extremely versatile technique for copying DNA. PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and real-time PCR (QPCR) which allow for quantitative measurement of DNA or RNA molecules. 36

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39. What does PCR need? Template (the DNA you are exploring) Sequence-specific primers flanking the target sequence, Forward & Reverse. Polymerases Nucleotides (dATP, dCTP, dGTP, dTTP) Magnesium chloride (enzyme cofactor) Buffer Water 38

40. PCR Requirements Magnesium chloride:.5-2.5mM Buffer: pH 8.3-8.8 dNTPs: 20-200µM Primers: 0.1-0.5µM DNA Polymerase: 1-2.5 units Target DNA:  1 µg 39

41. Steps in PCR Denaturation 94 to 95°C 1min Annealing 50 to 65°C 45sec Elongation 72°C 1-2min 40

42. How does PCR work? Heat (95-94 o C) to denature DNA strands Cool (50-65 o C) to anneal primers to template Warm (72 o C) to activate Taq Polymerase, which extends primers and replicates DNA Repeat multiple cycles 41

43. Denaturation Denaturation is the first step in PCR, in which the DNA strands are separated by heating to 95°C. The Hydrogen bonds between the two strands breaks down and the two strands separates. 42

44. Annealing Annealing is the process of allowing two sequences of DNA to form hydrogen bonds. The annealing of the target sequences and primers is done by cooling the DNA to 55°C. Usually time taken to anneal is 45 seconds. 43

45. Elongation Taq polymerase binds to the template DNA and starts adding nucleotides that are complementary to the first strand. This happens at 72°C as it is the optimum temperature for Taq Polymerase. 44

46. PCR Cycles 45

47. PCR Cycles 46

48. Denaturing Template Heat causes DNA strands to separate 3’ 5’ 3’ Denature DNA strands 95-94 o C 5’ 3’ 5’ 47

49. PCR Cycles 48

50. Annealing Primers Primers bind to the template sequence Taq Polymerase recognizes double-stranded substrate 3’ 5’ 3’ Primers anneal 65 o C 3’ 5’ 3’ 5’ 3’ 5’ 49

51. PCR Cycles 50

52. Taq Polymerase Extends 3’ 5’ 3’ 5’ 3’ 5’ Extend 72 o C 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Taq Polymerase extends primer DNA is replicated Repeat denaturing, annealing, and extending 30 cycles 51

53. PCR Cycles 52

54. The target product is made in the third cycle 3’ 5’ 3’3’ 3’3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Cycle 1 Cycle 2 Cycle 3 3’ 5’ 53

55. PCR Cycles Review Denaturation: 94°- 95°C Primer Annealing: 55°- 65°C Elongation of DNA: 72° Number of Cycles: 25-40 At 30 cycles there are 1,073,741,764 target copies (~1×10 9 ). 54

56. 55 PCR Analysis The process follows the principle of DNA replication

57. PCR Primers A primer is a strand of nucleic acid that serves as a starting point for DNA replication. These primers are usually short, chemically synthesized oligonucleotides A primer for each target sequence on the end of your DNA is needed. This allows both strands to be copied simultaneously in both forward and reverse directions. Replication starts at the 3'-end of the primer, and copies the opposite strand. 56

58. Primer Problems Primers should flank the sequence of interest Primers that match multiple sequences will give multiple products Repeated sequences can be amplified -but only if unique flanking regions can be found where primers can bind A primer may form a dimer with itself or with the other primer. 5´-ACCGGTAGCCACGAATTCGT-3´ |||||||||| 3´-TGCTTAAGCACCGATGGCCA-5´ 57

59. Primers That Form Hairpins Primers can have self-annealing regions within each primer (i.e. hairpin and foldbackloops) A primer may be self-complementary and be able to fold into a hairpin: 5´-GTTGACTTGATA ||||| T 3´-GAACTCT The 3´ end of the primer is base-paired, preventing it annealing to the target DNA. 58

60. PCR Taq DNA Polymerase Taq stands for Thermus aquaticus, which is a microbe found in 176°F hot springs in Yellow Stone National Forest. Taq DNA Polymerase (Taq Pol) is stable in high temperatures and acts in the presence of Mg. The optimum temperature for Taq Pol is 72°C 59

61. Disadvantages of Taq Pol Taq Pol lacks 3’ to 5’ exonuclease proof reading activity, commonly present in other polymerases. Taq mis-incorporates 1 base in 10 4. A 400 bp target will contain an error in 33% of molecules after 20 cycles. Error distribution will be random. 60

62. Limitations of PCR 61 Need for target DNA sequence information specially boundary regions of DNA to be amplified must be known. Primer Designing for unexplored ones. Infidelity of DNA replication. Taq Pol –Proof reading mechanism – Error 40% after 20 cycles Short size and limiting amounts of PCR product Up to 5kb can be easily amplified. Up to 40kb can be amplified with some modifications. Cannot amplify gene >100kb Cannot be used in genome sequencing projects.

63. How to overcome Difficulties? Pfu DNA Polymerase from Pyrococcus furiosus possesses 3' to 5' exonuclease proofreading activity. The error rate is only 3.5% after 20 cycles More amount of primer is added to avoid primer dimering. 62

64. Designing PCR Primers Primer sequences should be unique Primers should be ~20 bases long. The G/C content should be 45–55%. The annealing temperatures should be within 1°C of one another. The 3´-most base should be a G or C. The primers must not base pair with each other or with themselves or form hairpins. Primers must avoid repetitive DNA regions. 63

65. Advantages of PCR Speed Ease of use Sensitivity Robustness 64

66. Applications of PCR Screening human DNA samples for mutations associated with genetic diseases such as thalassemia and cystic fibrosis. PCR enables rapid amplification of template DNA for screening of uncharacterized mutations Can detect the presence of viral DNA before it turns in to a killer. Can obtain sequences from hair, blood stain, bones, other forensic specimens, other remains preserved at archaeological sites. 65

67. Applications of PCR 66 A common application of PCR is the study of patterns of gene expression. The task of DNA sequencing can also be assisted by PCR. PCR has numerous applications to the more traditional process of DNA cloning. An exciting application of PCR is the phylogenic analysis of DNA from ancient sources A common application of PCR is the study of patterns of genetic mapping PCR can also used in Parental testing, where an individual is matched with their close relatives.