Chapter 9_Molecular Techniques in medical science .pptx

molecular techniques

1. Molecular Technique By Abiy A. Abiy A. (BSc, MSc) 1 5/9/2024

2. Contents Recombinant DNA technology/genetic engineering Cloning Extraction of DNA and RNA from cells Gel electrophoresis Blotting PCR Abiy A. (BSc, MSc) 2 5/9/2024

3. What is recombinant DNA technology? Abiy A. (BSc, MSc) 3 5/9/2024 A technique mainly used to change the phenotype of an organism (host) when a genetically altered vector is introduced and integrated into the genome of the organism. So , basically, this process involves the introduction of a foreign piece of DNA structure into the genome which contains our gene of interest. This gene which is introduced is the recombinant gene and the technique is called the recombinant DNA technology.

4. Recombinant DNA Technology Using Recombinant DNA technology, we can isolate and clone single copy of a gene or a DNA segment into an indefinite number of copies . These new combinations of genetic material or (rDNA) molecules are introduced into the host cells, where they propagate and multiply This technique or methodology is called Recombinant DNA technology. Abiy A. (BSc, MSc) 4 5/9/2024

5. Recombinant DNA Technology DNA  molecules formed by laboratory methods of  genetic recombination  (such as  molecular cloning ) to bring together genetic material from multiple sources, Plasmid: a genetic structure in a cell that can replicate independently of the  chromosomes , typically a small circular DNA strand in the cytoplasm of a  bacterium  or  protozoan . Plasmids are much used in the laboratory manipulation of genes. Abiy A. (BSc, MSc) 5 5/9/2024

6. Abiy A. (BSc, MSc) 6 5/9/2024

7. Abiy A. (BSc, MSc) 7 5/9/2024 EcoRi (pronounced "eco R one") is a restriction endonuclease enzyme isolated from species E. coli . It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the restriction modification system. Recombinant DNA Technology…

8. Steps obtaining rDNA Step 1: Isolation of desired DNA Step 2: Isolation of vectors Step 3 : Production of chimeric DNA Step 4: Introduction of chimeric DNA in to Host cell Step 5: Multiplication and selection of cells with rDNA Step 6: Expression of gene products Abiy A. (BSc, MSc) 8 5/9/2024

9. 1. Isolation of DNA molecules The first step in making recombinant DNA is to isolate desired piece of donor DNA The procedure used for obtaining vector DNA depends on the nature of the vector:- Treat cells by enzyme and detergents Treat with phenol, protease and ribo -nuclease Ultracentrifugation Abiy A. (BSc, MSc) 9 5/9/2024

10. Isolation of DNA molecules… Treat with chilled ethanol Using restricted endonuclease enzyme cutting desired DNA molecules The desired DNA molecule is selected by electrophoresis or southern blotting techniques Finally we get pieces of sticky end DNA molecule. Sticky ends are overhangs of single-stranded DNA molecules after being cut with a restriction enzyme. Abiy A. (BSc, MSc) 10 5/9/2024

11. Cutting DNA The restriction enzymes EcoRi cuts a circular DNA molecule bearing one target sequence, resulting in a linear molecule with single stranded sticky ends. Abiy A. (BSc, MSc) 11 5/9/2024

12. 2. Isolation of vectors The structure of bacteria is surrounded by cell wall In order to get plasmid the bacteria cell wall is treated with enzyme, EDTA and lysozymes Then treated with sodium lauryl sarcosinate Ultracentrifugation Abiy A. (BSc, MSc) 12 5/9/2024

13. Ultracentrifugation A protocol for extracting plasmids DNA can be achieved by ultracentrifugation Plasmids DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient containing ethidium bromide The plasmid band is collected by punching a hole in the plastic centrifuge tube Abiy A. (BSc, MSc) 13 5/9/2024

14. Alkaline Lysis Another protocol relies on the observation that, at a specific alkaline pH, bacterial genomic DNA denatures but plasmids do not Subsequent neutralization precipitates the genomic DNA, but plasmids stay in solution By using restricted endonuclease enzymes finally we can get sticky end plasmids Abiy A. (BSc, MSc) 14 5/9/2024

15. 3. Formation of chimeric DNA The desired sticky end pieces of DNA molecules combined with sticky end plasmid with help of DNA ligase enzyme form chimeric /hybrid DNA molecules Ligase enzyme is the joining enzyme that join the vector DNA with the gene of interest this will produce the recombinant DNA Abiy A. (BSc, MSc) 15 5/9/2024

16. Conti… Restriction fragments from donor DNA are mixed with plasmid DNA and joined by their sticky ends, the initial attraction is due to the hydrogen bonds, but the sugar phosphate backbone is then joined using and enzyme called DNA ligase. Abiy A. (BSc, MSc) 16 5/9/2024

17. 4 . Introduction in to host cells Introduction of chimeric DNA in to host cells with the help of various gene transfer methods The most common host cells E. coli The vector is added to a flask containing a culture of E . coli . Calcium ions usually in the form of calcium chloride are added to the flask followed by a brief heat shock. This allows holes to briefly appear in the cell surface membrane of the E . coli making it permeable to DNA and allowing the plasmids to enter. Abiy A. (BSc, MSc) 17 5/9/2024

18. 5. Multiplication and selection of cells in the host cells Chimeric DNA multiply in the host cells and select by different methods like culture and fermentation tanks Abiy A. (BSc, MSc) 18 5/9/2024

19. 6. Expression of gene products The desired DNA express and produce a desired products like proteins, hormones, enzymes and vaccines etc. Abiy A. (BSc, MSc) 19 5/9/2024

20. Molecular tools in rDNA technology 1. Enzymes Exo-nuclease Endonuclease The most important one is restriction endonuclease (EcoRi) EcoRi- E. coli restriction I endonuclease, it is a molecular scissor. DNA ligase- DNA joining enzyme/molecular glue In needs ATP It forms Phosphodiester bonds Other enzymes like polymerase, alkaline phosphatase and reverse transcriptase are important in rDNA technology Abiy A. (BSc, MSc) 20 5/9/2024

21. Molecular tools in rDNA technology 2. Vectors There are vectors using in rDNA technology Plasmids- the most commonly used vector Eg Puc,pBR322 Bacteriophage- eg: phage λ vectors Cosmids- are hybrids between plasmid and phage λ vectors and clone large fragments of DNA . Eg : c2RB YAC-Yeast artificial chromosomes BAC-Bacterial artificial chromosomes MAC-Mammalian artificial chromosomes Abiy A. (BSc, MSc) 21 5/9/2024

22. Molecular tools in rDNA technology 3. Host cells Prokaryotic cells like E. coli and Bacillus subtilis Eukaryotic cells like fungi/yeast, plant cells and mammalian cells Abiy A. (BSc, MSc) 22 5/9/2024

23. Molecular tools in rDNA technology 4 . Gene transfer technique Transformation Transduction Electroporation Gene gun techniques Conjugation Abiy A. (BSc, MSc) 23 5/9/2024

24. 2.Cloning Abiy A. (BSc, MSc) 24 5/9/2024

25. Objective At the end of this portion you will be able to :- Define gene cloning? What are the required molecules for gene cloning? Explain different types of cloning? What are the Importance of cloning? Ethical issues in molecular biology 25 Abiy A. (BSc, MSc) 5/9/2024

26. Con… Cloning In genetics, to clone an organism is to make an exact copy of its DNA I s the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means In nature, some organisms produce clones through asexual reproduction eg Monozygotic (identical) twins Clone  Group of identical cells that share a common ancestry. Replicated DNA Abiy A. (BSc, MSc) 26 5/9/2024

27. Gene cloning Gene cloning   Is the process in which a  gene  of interest is located and copied ( cloned ) out of DNA extracted from an organism. These new combinations of genetic material or (rDNA) molecules are introduced into the host cells, where they propagate and multiply Also known as molecular cloning and recombinant DNA technology Abiy A. (BSc, MSc) 27 5/9/2024

28. Con.. Cloning is commonly done on small organisms, mostly plants, and on animals , such as the famous sheep called Dolly . Abiy A. (BSc, MSc) 28 5/9/2024

29. The molecules that are required for cloning are DNA to be cloned Cloning vector Enzymes ( cutting & Joining ) Abiy A. (BSc, MSc) 29 5/9/2024

30. Cloning vector DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell & produces many copies of itself & the foreign DNA A vector is any DNA molecule which is capable of multiplying inside the host to which our gene of interest is integrated for cloning. 30 Abiy A. (BSc, MSc) 5/9/2024

31. Some of Cloning Vectors Plasmids Phage Cosmids Bacterial Artificial Chromosomes (BAC) Yeast Artificial Chromosomes (YAC) 31 Abiy A. (BSc, MSc) 5/9/2024

32. Plasmid Abiy A. (BSc, MSc) 32 The most common used cloning vector 5/9/2024

33. Enzymes Restriction endonucleases and DNA ligases . In this process restriction enzyme function as scissors for cutting the DNA molecule The restriction enzymes EcoRi cuts a circular DNA molecule bearing one target sequence, resulting in a linear molecule with single stranded sticky ends Ligase enzyme is the joining enzyme that join the vector DNA with the gene of interest this will produce the recombinant DNA Abiy A. (BSc, MSc) 33 5/9/2024

34. Figure 20.3-3 Recombinant DNA molecule One possible combination DNA ligase seals strands DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. Restriction enzyme cuts sugar-phosphate backbones. Restriction site DNA 5  5  5  5  5  5  5  5  5  5  5  5  5  5  5  5  3  3  3  3  3  3  3  3  3  3  3  3  3  3  3  3  2 3 1 Sticky end GAATTC CTTAAG CTTAA G AATTC G G G AATTC CTTAA G G G G AATT C AATT C C TTAA C TTAA 34 Abiy A. (BSc, MSc) 5/9/2024 Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA.

35. Features of all cloning vectors P ermit the propagation Have a cloning site S electable markers for drug resistance 35 Abiy A. (BSc, MSc) 5/9/2024

36. The basic steps in a gene cloning Inserted into a circular DNA molecule The vector transports the gene into the host cell, With in the host cell the vector multiplies , Further vector replication takes place Get large number of cell divisions, a colony, or clone of identical host cells is produced. Which contains one or more copies of the recombinant DNA Abiy A. (BSc, MSc) 36 5/9/2024

37. Figure 20.2 Bacterium Bacterial chromosome Plasmid 2 1 3 4 Gene inserted into plasmid Cell containing gene of interest Recombinant DNA (plasmid) Gene of interest Plasmid put into bacterial cell DNA of chromosome (“foreign” DNA) Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of interest Protein expressed from gene of interest Protein harvested Copies of gene Basic research and various applications Basic research on protein Basic research on gene Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth 37 Abiy A. (BSc, MSc) 5/9/2024

38. Types of Cloning Therapeutic cloning Therapeutic cloning is the production of human embryos for research purposes. They are used to harvest stem cells that are used in human development research and potentially in the treatment of many diseases. 38 Abiy A. (BSc, MSc) 5/9/2024

39. Example of therapeutic cloning Cloning Insulin Insulin is a protein hormone produced in the pancreas that the body uses to regulate blood sugar concentrations Diabetics have lost the ability to produce insulin and must have an outside source of it In the 1920s, insulin from cows and pigs was isolated and made available to humans with diabetes. (Though it is not identical to human insulin) Supply was a major concern since the number of diabetics was on the rise Abiy A. (BSc, MSc) 39 5/9/2024

40. Cloning Insulin…. In 1978, Herbert Boyer and colleagues at the University of California in San Francisco created a synthetic version of human insulin using recombinant DNA technology The DNA sequence encoding/representing the instructions for growing/synthesizing insulin was separated and then inserted into the bacterium E. coli The E. coli then produced prodigious amounts of human insulin Abiy A. (BSc, MSc) 40 5/9/2024

41. Cloning Insulin…. Insulin Production The DNA for insulin is first isolated A plasmid made of DNA is removed from a bacterial cell A restriction enzyme cuts the plasmid DNA open, leaving sticky ends. The insulin gene, with complementary sticky ends is added . Abiy A. (BSc, MSc) 41 5/9/2024

42. Cloning Insulin…. DNA ligase enzyme splices (joins) together the plasmid and the insulin gene. The plasmid (now genetically modified) is inserted back into the bacterium. The bacterium host cell, divides and produces copies of the plasmid. The Bacterium makes human insulin using the gene in the plasmid. The insulin is extracted from the bacterial culture . Abiy A. (BSc, MSc) 42 5/9/2024

43. Joining DNA Abiy A. (BSc, MSc) 43 5/9/2024

44. Production of an Insulin Abiy A. (BSc, MSc) 44 5/9/2024

45. 2. Reproductive cloning Create an animal that has the same DNA as another animal. The famous Dolly the sheep was the first animal created by reproductive cloning. 45 Abiy A. (BSc, MSc) 5/9/2024

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47. 3. Recombinant DNA Is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together through the process of gene splicing In terms of genetic modification, it is created through the introduction of relevant DNA into an existing organismal DNA, such as the plasmids of bacteria Abiy A. (BSc, MSc) 47 5/9/2024

48. Abiy A. (BSc, MSc) 48 5/9/2024

49. 4 . DNA Cloning DNA cloning is the process of making multiple, identical copies of a particular piece of DNA Transfer of a DNA fragment of interest from one organism to a self-replicating genetic such as bacterial plasmid Plasmids self-replicating extra-chromosomal circular DNA molecules, distinct from normal bacterial genome 49 Abiy A. (BSc, MSc) 5/9/2024

50. DNA cloning Abiy A. (BSc, MSc) 50 5/9/2024

51. Use of Cloning : Gene therapy, Genetic engineering of organisms, and Genome sequencing. Getting an insight into an organism's genetic make-up and how this affects and influences the organism's life processes. 51 Abiy A. (BSc, MSc) 5/9/2024

52. U se… Solution for infertile couples Harvest embryonic stem cells Production of organs or tissues Develop new treatments for genetic-related disorders Genetic finger-printing Genetic engineer to create plant better nutrition value and resistance to disease 52 Abiy A. (BSc, MSc) 5/9/2024

53. U se… The improvement of reconstructive and cosmetic surgery C ure currently incurable diseases 53 Abiy A. (BSc, MSc) 5/9/2024

54. Disadvantage of cloning Losing gene diversity If gene diversity is lost due to excessive cloning, there are no mutations to allow some of the cloned group to survive a newly introduced disease . Ethical considerations One of these ethical concerns is that cloning is unnatural, and considered “playing God.” 54 Abiy A. (BSc, MSc) 5/9/2024

55. Ethical issues in Molecular biology There are ethical issues all the way along in biotechnology because human beings are capable of manipulating life as never before. Stem cell research raises the issue of where life begins and whether cells from a human embryo should be used for another human’s benefit. Abiy A. (BSc, MSc) 55 5/9/2024

56. CON… Present stem cell work concentrates on making regenerative cells for the cure of diseases But the possibility of cloning whole human beings has to be considered Dolly was cloned from a stem cell Abiy A. (BSc, MSc) 56 5/9/2024

57. Quiz What is recombinant DNA technology? (2 pts.) Define Plasmids (1 pts.) List molecular tools in rDNA technology (2 pts.) Abiy A. (BSc, MSc) 57 5/9/2024

58. 4. Extraction of DNA and RNA from cells Abiy A. (BSc, MSc) 58 5/9/2024

59. 4. Extraction of DNA and RNA from cells DNA extraction is a method to purify DNA by using physical and/or chemical methods from a sample separating DNA from cell membranes, proteins, and other cellular components The use of DNA isolation technique should lead to efficient extraction with good quantity and quality of DNA, which is pure and is devoid of contaminants, such as RNA and proteins Manual methods as well as commercially available kits are used for DNA extraction Various tissues including blood, body fluids, direct Fine needle aspiration cytology (FNAC) aspirate, formalin-fixed paraffin-embedded tissues, frozen tissue section, etc., can be used for DNA extraction Abiy A. (BSc, MSc) 59 5/9/2024

60. DNA extraction DNA extraction involves lysing the cells and solubilizing DNA, which is followed by chemical or enzymatic methods to remove macromolecules, lipids, RNA, or proteins DNA extraction techniques include O rganic extraction (phenol–chloroform method ) N onorganic method (salting out and proteinase K treatment ) and Kit methods Abiy A. (BSc, MSc) 60 5/9/2024

61. Three basic steps in DNA extraction Lysis Precipitation Purification Abiy A. (BSc, MSc) 61 5/9/2024

62. Lysis The nucleus and the cells are broken/open, thus releasing DNA This process involve mechanical disruption and uses lysozymes, extraction buffers, SDS and EDTA, phenol and /chloroforms, proteinase K to dissolve the cellular proteins and free DNA The purpose of extraction buffer Dissolve cellular membrane Assist in the removal of contaminants Abiy A. (BSc, MSc) 62 5/9/2024

63. Precipitation Separate the freed DNA from the cellular debris It involves use of sodium (Na+) ions to neutralize any negative charge in DNA molecules making them less water soluble and more stable Alcohol (eg isopropanol or ethanol) is then added, cause precipitation of DNA from the aqueous solution since it does not dissolve in alcohol Abiy A. (BSc, MSc) 63 5/9/2024

64. Purification After separation of DNA from aqueous solutions, it is then rinsed with alcohol Remove all the remaining cellular debris and unwanted materials Once the DNA is completely purified, it is usually dissolve in buffer Tris-EDTA (TE) again for convenient storage and handling Abiy A. (BSc, MSc) 64 5/9/2024

65. ORGANIC EXTRACTION Perhaps the most basic of all procedures in forensic molecular biology is the purification of DNA The key step, the removal of proteins, can often be carried out simply by extracting aqueous solutions of nucleic acids with phenol and/or chloroform Abiy A. (BSc, MSc) 65 5/9/2024

66. ORGANIC EXTRACTION Organic (phenol–chloroform) extraction uses  sodium dodecylsulfate  (SDS) and  proteinase K  for the enzymatic digestion of proteins and non-nucleic acid cellular components. A mixture of phenol:chloroform:isoamyl alcohol (25:24:1) is then added to promote the partitioning of lipids and cellular debris into the organic phase, leaving isolated DNA in the aqueous phase . Abiy A. (BSc, MSc) 66 5/9/2024

67. ORGANIC EXTRACTION Following centrifugation, the aqueous phase containing the purified DNA can be transferred to a clean tube for analysis. DNA can also be recovered and concentrated from the aqueous phase by ethanol precipitation or through the use of a centrifugal filter unit, which allows for additional purification and concentration of the DNA in the samples.   Abiy A. (BSc, MSc) 67 5/9/2024

68. PROCEDURE Cell Lysis Buffer - lyse cell membrane, nuclei are intact, pellet nuclei Resuspended nuclei, add Sodium Dodecyl Sulfate (SDS), Proteinase K. Lyse nuclear membrane and digest protein DNA released into solution is extracted with phenol-chloroform to remove proteinaceous material DNA is precipitated from the aqueous layer by the additional of ice cold 95% ethanol and salt Precipitated DNA is washed with 70% ethanol , dried under vacuum and re-suspended in TE buffer Abiy A. (BSc, MSc) 68 5/9/2024

69. ORGANIC EXTRACTION REAGENTS Cell lysis Buffer - Non-ionic detergent, Salt, Buffer , EDTA designed to lyse outer cell membrane of blood and epithelial cells, but will not break down nuclear membrane EDTA ( Ethylene di-amine tetra-acetic disodium salt ) is a chelating agent of divalent cations such as Mg2+. Mg2+ is a cofactor for Dnase nucleases . If the Mg2 + is bound up by EDTA, nucleases are inactivated. Abiy A. (BSc, MSc) 69 5/9/2024

70. ORGANIC EXTRACTION REAGENTS Proteinase K - it is usual to remove most of the protein by digesting with proteolytic enzymes such as Proteinase Proteinase K is approximately 10 fold more active on denatured protein. Proteins can be denatured by SDS or by heat Abiy A. (BSc, MSc) 70 5/9/2024

71. ORGANIC EXTRACTION REAGENTS Phenol/Chloroform - The standard way to remove proteins from nucleic acids solutions This procedure takes advantage of the fact that de- proteinization is more efficient when two different organic solvents are used instead of one Also, the final extraction with chloroform removes any lingering traces of phenol from the nucleic acid preparation Phenol is highly corrosive and can cause severe burns Abiy A. (BSc, MSc) 71 5/9/2024

72. Organic Extraction Advantage : Y ields relatively pure, high molecular weight DNA DNA is double stranded – good for RFLP Disadvantage: Time consuming Requires sample to be transferred to multiple tubes – increases risk of contamination Involves use of hazardous (and smelly !) chemicals Abiy A. (BSc, MSc) 72 5/9/2024

73. Non-Organic DNA Extraction Does not use organic reagents such as phenol or chloroform Digested proteins are removed by salting out with high concentrations of LiCl ( Lithium chloride) However, if salts are not adequately removed , n eutralize the Coulombic repulsion between the phosphate groups of these two strands thereby stabilizing the molecule in double stranded state. Problems could occur with the RFLP procedure due to alteration of DNA mobility (band shifting ). Abiy A. (BSc, MSc) 73 5/9/2024

74. Procedure 1. Cell Lysis Buffer - lyse cell membrane, nuclei are intact , pellet nuclei 2. Resuspend nuclei in Protein Lysis Buffer containing a high concentration of Proteinase K Lyse nuclear membrane and digest protein at 65oC for 2 hours Temperature helps denature proteins , and Proteinase K auto digests itself 3. To remove proteinaceous material, LiCl is added to a final concentration of 2.5 M, and incubated on ice. Proteins precipitate out and are pelleted by centrifugation. Abiy A. (BSc, MSc) 74 5/9/2024

75. Procedure 4. DNA remains in solution. Transfer supernatant to a new tube, care must be taken not to take any of protein pellet 5. DNA is precipitated by the addition of room temperature isopropanol. LiCl will not precipitate with DNA 6. Precipitated DNA is washed with 70% ethanol , dried under vacuum and re-suspended in TE buffer Abiy A. (BSc, MSc) 75 5/9/2024

76. RNA extraction What is RNA RNA = Ribonucleic acid • A type of nucleic acid with only one strand - ribose instead of de- oxyribose and using uracil instead of thymine (in DNA) • Provides the link between the genetic information through protein synthesis ( serve as template for protein synthesis) • Total RNA= rRNA (~85%), mRNA (~2%), tRNA and other molecules (~10 – 15 %) Abiy A. (BSc, MSc) 76 5/9/2024

77. The Purpose of RNA Extraction • Isolation of intact RNA is essential for many techniques used in gene expression analysis such as: Microarray analysis Northern blotting analysis cDNA library construction RT-PCR Abiy A. (BSc, MSc) 77 5/9/2024

78. RNA extraction Three ways to handle samples prior to RNA extraction Immediately disrupt the fresh samples Freeze the samples in liquid nitrogen Abiy A. (BSc, MSc) 78 5/9/2024

79. ORGANIC EXTRACTION Organic extraction (acidified phenol and chloroform) removes proteins, lipids, and DNA from the RNA sample RNA is then recovered by alcohol precipitation Advantage Can be use for large or small sample sizes Can modify extractions to remove high levels of fat and protein from samples Disadvantage Phenol and chloroform are hazardous Abiy A. (BSc, MSc) 79 5/9/2024

80. COLUMN PURIFICATION Glass filters bind the RNA while other cellular components are washed away • RNA is eluted in a highly purified form ADVANTAGES Rapid procedure No organic solvents required No alcohol precipitation needed Disadvantage Not as scalable as organic extraction methods Abiy A. (BSc, MSc) 80 5/9/2024

81. HOMOGENIZATION The first step in RNA extraction is to break down the cells or tissue so that the nucleic acids are released from the cells • Homogenization Tissue: mortar and pestle Cells: repetitive pipetting (re-suspend) • RNA extraction methods use a powerful chaotropic salt solution . This ‘lysis solution’ rapidly disrupts cells without destroying their nucleic acids • Total RNA isolation reagent (TRI reagent) as ‘lysis solution ’ combines phenol and guanidine thio-cyanate Abiy A. (BSc, MSc) 81 5/9/2024

82. PHASE SEPARATION Homogenate (from homogenization step ) must be store for 5 min at Room Temperature to permit the complete dissociation of nucleoprotein complexes • Chloroform used to: Separate solution in aqueous phase, interphase and organic phase RNA in aqueous phase, DNA ( interphase) and protein (organic phase ) • RNA, DNA and protein separation are based on density centrifugation • Centrifugation in COLD temperature ( 2 - 8°C). If perform at high temperature - a residual amount of DNA may mix in the aqueous phase Abiy A. (BSc, MSc) 82 5/9/2024

83. After separation Abiy A. (BSc, MSc) 83 5/9/2024

84. PHASE SEPARATION…. Why use chloroform?? Chloroform is organic solvent. So, lysed cell components that are hydrophobic will be trapped in these solvent ( eg : membrane lipids , hydrophobic polypeptide sequences ( protein) or polysaccharides etc.) Abiy A. (BSc, MSc) 84 5/9/2024

85. RNA PRECIPITATION Isopropanol: Precipitate RNA from the aqueous phase • RNA precipitate (often visible before centrifugation ) forms a gel-like or white-pellet on the side and bottom of the tube Abiy A. (BSc, MSc) 85 5/9/2024

86. RNA WASH & SOLUBILIZATION 75% ethanol: Wash RNA pellet away from any salt residual • RNase free water : Clean RNA re-suspended ( RNase free water) to ensure stability and long term storage Abiy A. (BSc, MSc) 86 5/9/2024

87. RNA QUALITY CONTROL Can be assessed by spectrophotometer ( eg: Nano-Drop ) • Ratio of the absorbance at 260 and 280 nm is used to assess the RNA purity Value 1.8-2.0 indicates that the RNA is pure If <1.8 : protein contamination > 2.0: solvent contamination Abiy A. (BSc, MSc) 87 5/9/2024

88. 5. GEL-ELECTROPHOROSIS Abiy A. (BSc, MSc) 88 5/9/2024

89. 5. GEL-ELECTROPHOROSIS Gel electrophoresis is an important technique for studying DNA that allows DNA fragments to be separated by size The gel acts as a kind of web for the DNA to move through Short fragments can move easily through the gel while long fragments get caught and so don’t move as far through the gel The DNA is pulled through the gel because it is negatively charged, as a result of its phosphate back bone The negatively charged DNA is attracted to the positive electrode at the end of the gel and so the DNA is pulled through the web like gel structure Abiy A. (BSc, MSc) 89 5/9/2024

90. GEL-ELECTROPHOROSIS Separates DNA fragments according to their size Abiy A. (BSc, MSc) 90 5/9/2024

91. GEL-ELECTROPHOROSIS Here is an example of what you might see on a gel. In reality we don’t see single strands because they are very small but we see bands on the gel made of lots of strands of the same size. You can see the longer strands are at the top of the gel, while the shorter strands have moved further down. Abiy A. (BSc, MSc) 91 5/9/2024

92. Materials and reagents For gel electrophoresis we need the following Casting tray Gel Combs Electrical supply DNA sample Loading dye Buffer solution: for better conductivity of electricity Ethidium bromides: Intercalating agents Abiy A. (BSc, MSc) 92 5/9/2024

93. Preparing the gel How do you prepare 1% agarose gel for electrophoresis? Pouring a Standard 1% Agarose Gel: Measure 1 g of agarose. Mix agarose powder with 100 mL 1xTAE in a microwavable flask. ... Microwave for 1-3 min until the agarose is completely dissolved (but do not over boil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. Abiy A. (BSc, MSc) 93 5/9/2024

94. Prepare the gel The gel has a “comb” in it. This is used when the gel is being poured so that it will have wells for the DNA to be put in when it sets. Abiy A. (BSc, MSc) 94 5/9/2024

95. Prepare the gel Carefully rest the pipette tip on the inside edge of the well. Add 20µL of each DNA sample+10µL loading dye into a well of the gel Fill all of the empty wells with 20 µL of water Abiy A. (BSc, MSc) 95 5/9/2024

96. Run the gel Run the gel for 100 V for 30 minutes Abiy A. (BSc, MSc) 96 5/9/2024

97. View the results Use the blue light Safe-Imager to view the DNA bands, even while it is running Use gel-documentation system Abiy A. (BSc, MSc) 97 5/9/2024

98. What to expect DNA Ladder This is a standard containing known lengths of DNA Abiy A. (BSc, MSc) 98 5/9/2024

99. What to expect.. In reality our bands might not be so clear but here is an example of what we expect to see The un-cut DNA is at the very top of the gel, that is because it is a very large piece and has struggled to make to through the gel very far The enzymes have cut the DNA into smaller pieces so they have multiple bands and some of them have made it further down the gel Abiy A. (BSc, MSc) 99 5/9/2024

100. What to expect.. Abiy A. (BSc, MSc) 100 5/9/2024

101. Blotting techniques What is blotting? Abiy A. (BSc, MSc) 101 5/9/2024

102. What is blotting? Blotting is the process of immobilization of sample nucleic acids on solid support The transfer of macromolecules from the gel to the membrane (either nitrocellulose or nylon membrane) Blots are techniques for transferring DNA, RNA and proteins on to a carrier so they can be separated, and often follows the use of a gel electrophoresis. Abiy A. (BSc, MSc) 102 5/9/2024

103. TYPES OF BLOTTING TECHNIQUES Blotting technique Southern Blot It is used to detect DNA. Northern Blot It is used to detect RNA. Western blot It is used to detect protein. Abiy A. (BSc, MSc) 103 5/9/2024

104. SOUTHERN BLOTTING The technique was developed by E.M. Southern in 1975 The Southern blot is used to detect the presence of a particular piece of DNA in a sample Is a technique that combines the use of restriction enzymes, electrophoresis and DNA probes to generate, separate and detect pieces of DNA Abiy A. (BSc, MSc) 104 5/9/2024

105. Cont…. Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size separated DNA to a filter membrane for probe hybridization The key to this method is DNA-DNA Hybridization Hybridization - Process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA Abiy A. (BSc, MSc) 105 5/9/2024

106. Steps in southern blotting 1.DNA digestion by restriction enzyme 2.DNA gel electrophoresis 3.Blotting –capillary action 4.Hybridization and washing-adding probes 5.Detection-Autoradiography or fluorescence Abiy A. (BSc, MSc) 106 5/9/2024

107. Southern blotting Abiy A. (BSc, MSc) 107 5/9/2024

108. APPARATUS Capillary plotting apparatus Abiy A. (BSc, MSc) 108 5/9/2024

109. Steps in southern blotting 1. Digest the DNA with an appropriate restriction enzyme. 2.The complex mixture of fragments is subjected to gel electrophoresis to separate the fragments according to size. Abiy A. (BSc, MSc) 109 5/9/2024

110. Cont…. 3 . The restriction fragments present in the gel are denatured with alkali and transferred onto a nitrocellulose filter or nylon membrane by blotting. This procedure preserves the distribution of the fragments in the gel, creating a replica of the gel on the filter. Abiy A. (BSc, MSc) 110 5/9/2024

111. Cont…. 5 . The filter is incubated under hybridization conditions with a specific radiolabeled DNA probe. The probe hybridizes to the complementary DNA restriction fragment.  Abiy A. (BSc, MSc) 111 5/9/2024

112. Cont…. 6 . Excess probe is washed away and the probe bound to the filter is detected by autoradiography, which reveals the DNA fragment to which the probe hybridized. Abiy A. (BSc, MSc) 112 5/9/2024

113. Abiy A. (BSc, MSc) 113 5/9/2024

114. SUMMARY OF PROCEDURE 1. Extract and purify DNA from cells 2. DNA is restricted with enzymes 3. Sort by electrophoresis 4. Denature DNA 5. Transfer to nitrocellulose paper 6. Block with excess DNA 7. Wash off unbound probe 8. Autoradiograph Abiy A. (BSc, MSc) 114 5/9/2024

115. APPLICATIONS To detect mutant gene Useful for forensic purpose (paternity, identifying thieves, and rapists) It is used for DNA fingerprinting, preparation of RFLP maps Confirmation of DNA cloning results Abiy A. (BSc, MSc) 115 5/9/2024

116. Northern Blotting Northern blotting is a technique for detection of specific RNA sequences Northern blotting was developed by James Alwine and George Stark at Stanford University (1979) and was named such by analogy to Southern blotting There will be RNA-DNA hybridization Abiy A. (BSc, MSc) 116 5/9/2024

117. Steps involved in Northern blotting 1. RNA is isolated from several biological samples (e.g. various tissues, various developmental stages of same tissue etc.) * RNA is more susceptible to degradation than DNA. Abiy A. (BSc, MSc) 117 5/9/2024

118. Cont…… 2. Sample’s are loaded on gel  and the RNA samples are separated according to their size on an agarose gel . The resulting gel following after the electrophoresis run.   Abiy A. (BSc, MSc) 118 5/9/2024

119. Cont…… 3. The gel is then blotted on a nylon membrane or Aminobenzyloxymethyl papers by creating the sandwich arrangement Abiy A. (BSc, MSc) 119 5/9/2024

120. Cont…… 4. The membrane is placed in a dish containing hybridization buffer with a labeled probe Thus, it will hybridize to the RNA on the blot that corresponds to the sequence of interest  5. The membrane is washed to remove unbound probe Abiy A. (BSc, MSc) 120 5/9/2024

121. Cont…… 6. The labeled probe is detected via autoradiography or via a chemi -luminescence reaction (if a chemically labeled probe is used).  In both cases this results in the formation of a dark band on an X-ray film. Now the expression patterns of the sequence of interest in the different samples can be compared Abiy A. (BSc, MSc) 121 5/9/2024

122. Abiy A. (BSc, MSc) 122 5/9/2024

123. APPLICATIONS A standard for the study of gene expression at the level of mRNA ( messenger RNA transcripts) Detection of m RNA transcript size Study RNA degradation Study RNA splicing Study RNA half-life Abiy A. (BSc, MSc) 123 5/9/2024

124. Western blotting Western blotting (1981) is an Immunoblotting technique which rely on the specificity of binding between a protein of interest and a probe (antibody raised against that particular protein) to allow detection of the protein of interest in a mixture of many other similar molecules Use of antibody probes The SDS PAGE technique is a prerequisite for Western blotting Proteins separate based on their charge, size/shape and mass Abiy A. (BSc, MSc) 124 5/9/2024

125. Steps in western blotting A protein sample is subjected to electrophoresis on an SDS-polyacrylamide gel, protein separated based on charge, size/shape and mass Electro-blotting transfers the separated proteins from the gel to the surface of a nitrocellulose membrane . Abiy A. (BSc, MSc) 125 5/9/2024

126. Cont… 3 . The blot is incubated with a generic protein (such as milk proteins) which binds to any remaining sticky places on the nitrocellulose-blocking step 4. Probing :- a n antibody that is specific for the protein of interest (the primary antibody - Ab1) is added to the nitrocellulose sheet and reacts with the antigen.  Only the band containing the protein of interest binds the antibody, forming a layer of antibody molecules . Primary antibody is not a reporter Abiy A. (BSc, MSc) 126 5/9/2024

127. Cont… 5. After washing for removal of non-specifically bound Ab1, second antibody (Ab2 ) is added, which specifically recognizes  the Fc domain of the primary antibody and binds it. Ab2 is radioactively labeled, or is covalently linked to a reporter enzyme, which allows to visualize the protein-Ab1-Ab2 complex. Secondary antibody is a reporter Anti primary antibody Abiy A. (BSc, MSc) 127 5/9/2024

128. Abiy A. (BSc, MSc) 128 5/9/2024

129. Application 1. The confirmatory for HIV test 2. Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE( 3. Some forms of Lyme disease testing employ Western blotting. Abiy A. (BSc, MSc) 129 5/9/2024

130. Dot blotting (Reading Assignment) Used for detecting DNA/RNA Modification of southern and Northern blotting techniques Abiy A. (BSc, MSc) 130 5/10/2024

131. Polymerase Chain Reaction (PCR) Abiy A. (BSc, MSc) 131 5/9/2024

132. Define Polymerase chain reaction Explain stages of PCR Describe PCR according to time & temperature termed steps. Discuss PCR components Explain different types of PCR. Objectives Abiy A. (BSc, MSc) 132 5/9/2024

133. P olymerase C hain R eaction (PCR) S pecific sequences within a DNA or cDNA template can be copied, or “amplified”, many thousand- to a million-fold using sequence specific oligonucleotides, heat stable DNA polymerase, and thermal cycling. Introduction Abiy A. (BSc, MSc) 133 5/9/2024

134. Introduction….. Abiy A. (BSc, MSc) 134 5/9/2024

135. PCR is used widely in; Molecular cloning Pathogen detection Genetic engineering Mutagenesis Genetics, producing molecular markers Forensics Introduction…. Abiy A. (BSc, MSc) 135 5/9/2024

136. Denaturation Annealing and Extension Each of these steps is repeated at 30–40 times, termed cycles Stages in the PCR Abiy A. (BSc, MSc) 136 5/9/2024

137. S tages in PCR …. Abiy A. (BSc, MSc) 137 5/9/2024

138. Abiy A. (BSc, MSc) 138 5/9/2024

139. Abiy A. (BSc, MSc) 139 5/9/2024

140. PCR components Abiy A. (BSc, MSc) 140 5/9/2024

141. DNA template Contains region to be amplified Any DNA desired Should be free of polymerase inhibitors Primers Specific for ends of amplified region Forward and Reverse Depends on primer length, GC content, etc. Length 15-30 nucleotide PCR components Abiy A. (BSc, MSc) 141 5/9/2024

142. Nucleotides dNTPs - deoxynucleotidetriphosphates : DNA building blocks. Added to the growing chain dATP , dGTP , dCTP , dTTP Buffer Stabilizes the DNA polymerase, DNA, and nucleotides E g Triton X-100 or Tween Mg ++ ions Essential co-factor of DNA polymerase Enhances enzymatic activity of DNA polymerase PCR components …. Abiy A. (BSc, MSc) 142 5/9/2024

143. DNA Polymerase The enzyme that does the extension TAQ polymerase or similar Heat-stable PCR components …. Abiy A. (BSc, MSc) 143 5/9/2024

144. PCR machine Abiy A. (BSc, MSc) 144 5/9/2024

145. PCR STEPS Abiy A. (BSc, MSc) 145 5/9/2024

146. PCR STEPS….. Abiy A. (BSc, MSc) 146 5/9/2024

147. PCR STEPS….. Abiy A. (BSc, MSc) 147 5/9/2024

148. PCR STEPS….. Abiy A. (BSc, MSc) 148 5/9/2024

149. PCR STEPS…… The mixture is heated again at 90-95 ℃ to denature the molecules and separate the strands and the cycle repeated. Each new strand then acts as a template for the next cycle of synthesis. Abiy A. (BSc, MSc) 149 5/9/2024

150. Post amplification detection The amplification product can be detected using gel electrophoresis followed by ethidium bromide staining and visualization with UV transillumination. PCR STEPS…. Abiy A. (BSc, MSc) 150 5/9/2024

151. Modifications and different types of PCR There are different types of PCR C onventional PCR Real time PCR Advanced PCR Abiy A. (BSc, MSc) 151 5/9/2024

152. Traditional PCR has advanced from detection at the end-point of the reaction to detection while the reaction is occurring C onventional PCR is not quantitative, but rather qualitative Abiy A. (BSc, MSc) 152 Conventional PCR 5/9/2024

153. C onventional PCR Abiy A. (BSc, MSc) 153 5/9/2024

154. I s the technique of collecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step Allows real time monitoring of PCR amplification products by use of fluorescence In real-time quantitative PCR, PCR product is measured at each cycle. Real time PCR Abiy A. (BSc, MSc) 154 5/9/2024

155. Real time PCR….. Abiy A. (BSc, MSc) 155 5/9/2024

156. SYBR Green is the most commonly used fluorescent dye It binds specifically to double-stranded DNA . that it allowing measurement of the amount of PCR product Abiy A. (BSc, MSc) 156 Function of Syber green in PCR 5/9/2024

157. Taq-Man PCR is a type of real-time PCR and uses a nucleic acid probe complementary to an internal segment of the target DNA The probe is labeled with two fluorescent moieties. The emission spectrum of one overlaps the excitation spectrum of the other, resulting in “quenching” of the first fluorophore by the second Abiy A. (BSc, MSc) 157 Taq-Man real time PCR 5/9/2024

158. Real time PCR….. Abiy A. (BSc, MSc) 158 5/9/2024

159. The main difference between SYBR Green and Taqman is that: SYBR green is a dsDNA binding dye used to detect PCR products accumulated during the PCR reaction whereas Taqman is a fluorogenic probe specific to a target gene, which accumulates during PCR Abiy A. (BSc, MSc) 159 SYBR Green and Taqman 5/9/2024

160. Abiy A. (BSc, MSc) 160 5/10/2024

161. Reverse Transcriptase-PCR RT-PCR similar to PCR except it allows amplification of small amount of ribonucleic acid (RNA) It is used to detect viruses with an RNA genome and to detect RNA transcripts RT-PCR is a two-step procedure that involves making a cDNA copy of the mRNA. Reverse transcription followed by PCR allows cloning of genes starting from the mRNA Advanced PCR Abiy A. (BSc, MSc) 161 5/9/2024

162. The principle is to convert RNA into its complementary DNA sequence by reverse transcriptase, to synthesis a second strand with DNA polymerase, and finally to generate a ds cDNA molecule which can be amplified by PCR in the normal way Abiy A. (BSc, MSc) 162 Principle of RT-PCR 5/9/2024

163. RT-PCR….. Abiy A. (BSc, MSc) 163 5/9/2024

164. S mall amount of the first PCR is used for a second PCR amplification with a new set of internal primers The use of a second round of PCR with a new set of internal primers is called a nested PCR Nested PCR therefore increase the sensitivity as much as 1000-fold compared with conventional PCR and increase specificity, but the techniques require more work and longer assay times and carry an increased risk of contamination. Nested PCR Abiy A. (BSc, MSc) 164 5/9/2024

165. Nested PCR…. Abiy A. (BSc, MSc) 165 5/9/2024

166. PCR represents the prototype of nucleic acid amplification techniques Today, PCR is being used in research as well as in laboratories, clinics, and hospitals for genetic tests, and in forensic laboratories for the analysis of DNA evidence. S ummary Abiy A. (BSc, MSc) 166 5/9/2024

167. The PCR consists of three defined sets of times and temperatures termed steps: denaturation, annealing and extension . All PCR really requires in the way of equipment is a reaction tube, reagents, and a source of heat. S ummary ….. Abiy A. (BSc, MSc) 167 5/9/2024

168. There are Modifications and different types of PCR such as RT-PCR, Real time PCR and Nested PCR. S ummary … . Abiy A. (BSc, MSc) 168 5/9/2024

169. Thank you Abiy A. (BSc, MSc) 169 5/9/2024