Chapter 16. Just do it! Techniques of molecular biology. Prepared by Woojoo Choi

Transcription

1 Chapter 16. Just do it! Techniques of molecular biology Prepared by Woojoo Choi

2 Gel electrophoresis 1) Gel Electrophoresis movement of charged molecules toward an electrode of the opposite charge Used to separate nucleic acids or proteins or any molecule that has a charge Agarose gel electrophoresis can be used to purify DNA or it can be used to measure the size of fragments. 2

3 Gel electrophoresis Fragments of DNA have the same number of charges per unit length. They all cruise along at the same speed toward the positive electrode. 3

4 Gel electrophoresis To separate the fragments we run them through a gel. Gelatin sets due to a microscopic meshwork formed by its own protein fibers. Gel for DNA work are made of agarose and gel for protein work are made of polyacrylamide. 4

5 Gel electrophoresis The larger molecules find it more difficult to squeeze through the gaps but the smaller ones are slowed down much less. The result is that the DNA fragments separate in order of size. 5

6 Gel electrophoresis Both agarose and DNA are naturally colorless, so cannot see where the DNA has ended up. To find DNA fragments, ethidium bromide (Etbr), a dye that stains DNA and RNA when viewed under UV light, is used. After the bands are located, they are cut out of the agarose slab and the DNA is extracted to yield a pure fragment. To find the size of DNA, we run a set of standard DNA fragments of known sizes alongside, on the same gel. 6

7 Gel electrophoresis In the case of protein, depending on its overall amino acid composition, a protein may be positive, negative or neutral. To avoid these complication, proteins are boiled in a solution of the detergent. Sodium dodecyl sulfate (SDS): a detergent used to unfold proteins 7

8 Gel electrophoresis 8

9 Gel retardation and footprinting 1) Which regulatory proteins affect the gene in question? 2) This means measuring whether or not the regulatory protein binds to the regulatory region in front of the gene. 3) To test this, we need purified DNA and purified regulatory protein. 9

10 Gel retardation and footprinting 4) Gel retardation (band shift): When a protein binds to a segment of DNA, the DNA will move slower through a gel and the DNA band will be shifted to a new position. 10

11 Gel retardation and footprinting 5) Footprint: When a protein binds to DNA at a specific site it can protect the DNA from being cut in this region. 6) The result may be visualized as a missing group of bands when the DNA is run on a gel. 11

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13 Measuring DNA and RNA with ultraviolet light 1) If a beam of UV light is shone through a solution containing nucleic acids, the proportion of the UV absorbed depends on the amount of DNA or RNA. 13

14 Measuring DNA and RNA with ultraviolet ligh 2) The UV light is actually absorbed by the aromatic rings of the bases. 3) In a solution of unlinked nucleotides, the bases are more spread out. 4) In a DNA double helix, the bases are stacked on top of each other so that relatively less UV light is absorbed. 5) In RNA that is single stranded, the situation is intermediate. 6) UV absorbance: DNA < RNA < free nucleotides 14

15 Radioactive labeling 1) One matter we need to tackle is the question of how to detect and measure DNA or RNA when it binds to a filter or runs on a gel. 2) We also need to distinguish the DNA by its source. 3) Originally labeling the DNA was done by making the DNA to be traced radioactive. Radioactive: emitting radiation due to unstable atoms that break down releasing α-, β-, or γ-rays 15

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17 Radioactive labeling 4) Radioisotopes are the radioactive forms of an element. 5) Two are especially important: the radioactive isotopes of phosphorus, 32 P; and sulfur, 35 S 17

18 Radioactive labeling 18

19 Radioactive labeling 6) 35 S is usually preferable to 32 P in most molecular biology applications. 7) There are two reasons The half life of 35 S is 88 days whereas 32 P is 14 days, so it does not all disappear so fast. The radiation emitted by 35 S is of lower energy than for 32 P. 8) What this means in practice is that the radiation does not travel so far, so the radioactive bands are more precisely located and not so fuzzy. It is more accurate. 19

20 Radioactive labeling 9) Two methods most widely used to measure radioactivity scintillation counting: detection and counting of individual microscopic pulses of light If our sample is in liquid or on a strip of filter paper, we use scintillation counting. autoradiography: allowing radioactive materials to take pictures of themselves by laying them flat on photographic film If our sample is flat, a gel or a blot, we use autoradiography. 20

21 Scintillation counting 1) Scintillation counter: machine that detects and counts pulses of light 2) If β-particles are absorbed by special chemicals called scintillants, they result in the emission of a flash of light. 3) The light pulses are detected by a photocell. 21

22 Scintillation counting 4) Scintillation counter can also be used to measure light generated by chemical reactions. 5) In this case, the light is emitted directly so no scintillant fluid is needed and the luminescent sample is merely inserted directly. 22

23 1) The gel or filter is dried. Autoradiography 2) A sheet of photographic film is laid on top of the gel or filter and left for several hours. 3) The film will be darkened where the radioactive DNA bands or sports are found. 4) Exposing the film must be carried out in a dark room to avoid visible light. 23

24 Non-radioactive detection 1) Although the low level of radioactivity is of little hazard, the massive burden of ever-increasing government regulation has made it relatively cheaper and quicker to use other detection methods. 24

25 Non-radioactive detection 2) Fluorescene: when a molecule absorbs light of one wavelength and then emits light of another, longer wavelength of lower energy 25

26 Non-radioactive detection 3) Fluorescent dyes can be attached to DNA molecules and modern automated methods for DNA sequencing make use of such fluorescent tagging (see Ch. 22). 4) In this case, the dyes are zapped by a laser and then emit light of a longer wavelength than the laser. 26

27 Non-radioactive detection 5) Another instrument is used. Fluorescence activated cell sorter (FACS): machine that sorts particles, such as cells or chromosomes, according to their fluorescence 27

28 Non-radioactive detection 6) Biotin (vitamin) and digoxigenin (steroid molecule from the foxglove plants) are two tags widely used for labeling DNA. 28

29 Non-radioactive detection 7) Molecular biologists use avidin (protein found in egg whites and used for the defense mechanism) to bind the biotin tag. 29

30 Non-radioactive detection 8) Attached to the back side of the avidin is another molecule that provides the actual detection system. 9) Digoxigenin is used similarly. 10)In this case, an antibody that recognizes and binds to the digoxigenin is used. 30

31 Non-radioactive detection 11)What is the detection system? 1 An enzyme that generates a colored product may be attached to the avidin or antibody. 31

32 Non-radioactive detection One good example is alkaline phosphatase. alkaline phosphatase: an enzyme that chops phosphate groups from a wide range of molecules X-phos: substance split by alkaline phosphatase, so yielding a blue dye 32

33 Non-radioactive detection 2 An enzyme which produces light by a chemical reaction may be used instead. Chemiluminescence: emission of light as a side product of a chemical reaction 33

34 Restriction fragment length polymorphisms (RFLPs) 1) If we change even a single base within this sequence, we will prevent the enzyme from cutting the DNA. 34

35 Restriction fragment length polymorphisms (RFLPs) 2) Restriction fragment length polymorphisms (RFLPs): differences in lengths of fragments made by cutting the DNA with restriction enzyme 3) It may be used to identify organisms or analyze relationships. 4) If we examine the same region of a chromosome from two related, though not identical, we find that the DNA sequence is similar but not quite the same. 5) Consequently, restriction sites that are present in one version of a sequence may be missing in its relatives. 6) If we cut up two related but different DNA molecules with the same restriction enzyme, we may get segments of different lengths. 35

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37 Hybridization of DNA and RNA 1) Melting: when used in reference to DNA, refers to its separation into two strands as a result of heating 2) Melting temperature: the temperature at which the two strands of a DNA molecule are half-way unpaired 3) Denaturation: In the case of DNA, this amounts to separation into two strands. 37

38 Hybridization of DNA and RNA 4) GC base pair has three hydrogen bonds and AT has two hydrogen bond. GC base pairs are stronger than AT base pairs. As the temperature rises, AT pairs come apart first. The regions of DNA with lots of GC base pairs hold on longer. 38

39 Hybridization of DNA and RNA Melting temperature 39

40 Hybridization of DNA and RNA 5) Hybrid DNA: artificial double stranded DNA formed by two single strands from two different sources 40

41 Hybridization of DNA and RNA 6) The formation of hybrid DNA has a wide variety of uses. 7) For example, we can test how closely two DNA are related. First, DNA molecules are melted and attached to a filter. 41

42 Hybridization of DNA and RNA Then, we take the second sample and pour the solution through the filter. The more closely related our two molecules are, the more hybrid molecules will be formed and the higher the proportion of DNA 2 which will be bound by filter. 42

43 Hybridization of DNA and RNA 8) Example of using this technique (Cloning by hybridization). This approach allows us to isolate new genes as long as we already have a known gene closely related enough in sequence to hybridize well. 43

44 Southern, Northern and Western blotting 1) Southern blotting: hybridization technique in which DNA binds to DNA - named Edward Southern, its inventor 44

45 Southern, Northern and Western blotting 2) Northern blotting: hybridization technique in which a DNA probe binds to an RNA target molecule 3) Western blotting: detection technique in which a probe, usually an antibody, binds to a protein target molecule 4) South-Western blotting: detection technique in which a dsdna probe binds to a protein target molecule 45

46 Southern, Northern and Western blotting 46

47 Zoo blotting 1) The coding sequences are what we want, yet they are only a small fraction of the total DNA. 2) How do we identify a coding region? During evolution, non-coding DNA mutates and changes rapidly, whereas coding sequences change much more slowly and can still be recognized after very long time between two species (see Ch. 23). 3) Zoo blotting comparative Southern blotting using DNA target molecules from several different animals to test whether the probe DNA is from a coding region 47

48 Zoo blotting If a DNA is from coding sequence, it will probably hybridize with some fragment from most other closely related animals. If not, it will probably hybridize only to the human DNA. 48

49 Chromosome walking 1) Positional cloning: any cloning procedure based on knowing a gene s location rather than its function 2) One of the simplest version of this is chromosome walking. 3) Chromosome walking: method for cloning neighboring regions of a chromosome by successive cycles of hybridization using overlapping probes 4) In practice, this approach is used when we already have one cloned segment of DNA and we think that neighboring genes might be of interest. 49

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53 Subtractive hybridization 1) Removal of unwanted genes by hybridization, leaving behind the gene being sought 2) Suppose that we have a chromosome with a certain sequence of DNA missing. 3) Since we want to find the missing DNA, the corresponding chromosome from a healthy individual is obtained. 53

54 Subtractive hybridization 4) Cutting out our two chromosomes into fragments of convenient size. 5) Hybridize these two batches of DNA. 6) We would get hybrid molecules for all regions of the DNA except the region of the deletion which is present only in the healthy chromosome. 7) In this way, we have subtracted out all the segments of DNA that we do not want. 54

55 Subtractive hybridization 8) If we want to extract the mrna, we use hybridization. 9) Since mrna ends in a poly-a tail, we make a piece of poly T. 10)The poly-a tails hybridize to the poly-t and the mrna is trapped. 55

56 Subtractive hybridization 56

57 Subtractive hybridization 57

58 A cdna library is a collection of DNA, sans introns 1) cdna library a collection of cloned genes present as their complementary DNA versions and carried on an appropriate plasmid or virus vector - cdna form in eukaryotic genes (lacking introns) 58

59 FISH -Fluorescence in situ hybridization 1) Fluorescence in situ hybridization (FISH) using a fluorescent tagged probe to see a molecule of DNA or RNA in its natural location 59

60 FISH -Fluorescence in situ hybridization 2) Application of FISH 1 Find the cell which contain virus genes and the location of the virus gene. 60

61 FISH -Fluorescence in situ hybridization 2 Find which chromosome carries gene and localize the gene to a region on the chromosome. 61

62 FISH -Fluorescence in situ hybridization 3 Detect mrna within our target tissue since one of the two strands of the DNA will bind to the RNA. 62

63 Reporter gene 1) Reporter gene: a gene that is easy to detect and which is inserted for diagnostic purposes 1 Antibiotic resistance gene: gene conferring resistance to an antibiotic 63

64 Reporter gene 2 LacZ gene: the gene that encodes β-galactosidase which split lactose 64

65 Artificial compounds of galactose ONPG (o-nitrophenyl galactoside): substance split by β- galactosidase and yielding yellow o-nitrophenol X-gal: substance split by β-galactosidase and yielding a blue dye 65

66 3 phoa gene: the gene that encodes alkaline phosphatase Alkaline phosphatase: enzyme that splits off phosphate groups from many different molecules (eg, o-nitrophenyl phosphate, X- phos, Lumi-phos) 4 A more sophisticated reporter gene encodes for luciferase. Lux gene (from bacteria) and Luc gene (from animals) 66

67 Gene fusion 1) Reporter genes can be used to track the physical location of a segment of DNA or for more detailed genetic analysis. 2) Gene fusion: hybrid in which the regulatory sequences from one gene are joined to the coding region of another gene 67

68 3) The gene fusion will be controlled the same way that the target gene was controlled. 4) Instead of making the original gene product, it makes the enzyme belonging to the reporter gene. 5) We can monitor gene expression quickly and easily under a zillion different conditions by assaying the reporter gene enzyme. 68

69 Chemical synthesis of DNA 1) Nucleotides are added one by one and the growing strand of DNA remains attached to the glass beads until the synthesis is complete. 2) The problem is that each deoxynucleotide has two hydroxyl groups, one for bonding to the next nucleotide and the other for bonding to the previous nucleotide. 3) So each time a nucleotide is added, we must first block one of its hydroxyl groups and activate the one we want to react. 69

70 Chemical synthesis of DNA 4) Synthesis: 3 to 5 direction (5 to 3 direction in the cell) 70

71 Chemical synthesis of DNA 71

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73 The electron microscope 1) With an ordinary light microscope, objects down to almost a micron in size can be seen. 2) Typical bacteria are a micron or two long by about half a micron wide. 3) Although they are visible under a light microscope, the internal details are too small to make out. 4) The resolving power of a microscope depends on the wavelength of the light. 73

74 The electron microscope 5) A beam of electrons has a much smaller wavelength than does visible light and can distinguish detail far beyond the limits of resolution by light. 6) It allows to visualize the layers of the bacterial cell wall and to see the chromosome. 7) Because electrons are easily absorbed, even by air, the electron beam is used inside a vacuum chamber and the sample must be sliced extremely thin. 8) To improve contrast, cell components are usually stained with compounds of heavy metals (uranium, osmium or lead) of which strongly absorb electrons. 74

75 The electron microscope 9) Uncoiled DNA molecules can be seen if they are shadowed with metal atoms (gold, platinum, or tungsten) to increase the absorption of electrons. 75

76 The electron microscope 10)An example of this approach was the direct visualization of the introns. R-loop analysis: when a DNA copy of a gene is base paired with the corresponding mrna, the extra regions in the DNA, which have no partners in the mrna, appear as loops. The resulting loops can be directly seen under the electron microscope. 76

77 X-ray crystallography 1) X-ray crystallography (X-ray diffraction): determination of 3-D crystal structure by using X-rays 2) When a beam of X-rays is shone through a substance, the X-rays are scattered by the atoms they encounter. 3) If the target substance is a crystal with a regular structure, the scattering of the X-rays will give rise to a regular diffraction pattern, though complex. Diffraction pattern: array of spots formed by X-rays after traveling through a crystal 4) In practice, the crystal is rotated into a variety of positions on a computer-controlled stage. 5) The diffraction patterns are recorded and, after computer analysis, are used to generate a 3-D atomic map of the protein molecule. 77

78 X-ray crystallography 78