Genetic Engineering for C&I PDF

Genetic Engineering For Computers & Information Students Prepared by Dr. Samah Shehata Lecturer of Biochemistry  ‫❖ الرؤية‪:‬‬ ‫تسعى كلية الصيدلة جامعة الفيوم إلى التميز في مجال...

Genetic Engineering For Computers & Information Students Prepared by Dr. Samah Shehata Lecturer of Biochemistry  ‫❖ الرؤية‪:‬‬ ‫تسعى كلية الصيدلة جامعة الفيوم إلى التميز في مجال التعليم الصيدلي والبحث العلمي‬ ‫التطبيقي الخدمي على المستوى المحلى واإلقليمي‪.‬‬ ‫❖ الرسالة‪:‬‬ ‫تهدف كلية الصيدلة جامعة الفيوم الى إمداد سوق العمل بصيادلة ذوي كفاءة مهنية‬ ‫وأخالقية عالية‪.‬كما تسعى الكلية لتحقيق مستوى عالي وتنافسي في مجال البحث‬ ‫العلمي وأيضا في مجال المشاركة المجتمعية‪.‬‬ ‫]‪[2‬‬  GENETICS Introduction to biotechnology Biotechnology is the broad area of biology involving living systems and organisms to develop or make products. It is "Any technological application that uses biological systems, living organisms, or derivatives to make, modify products or processes for specific use". It is "The use of microorganisms, plants, and animals or their derivatives for the production of useful compounds, thus improve human health and environment". The historical uses of biotechnology: Food Production: Food biotechnology encompasses genetic engineering approaches to enhance food quality by microorganisms, animals, or plants; microbial fermentation processes for manufacturing innovative food, enzyme, and additives; the introduction of new technologies for food packaging and preservation. Bread Yogurt Cheese Manufacture of beer, wine (Fermentation) To make the alcohol, the sugars is first released from grain of choice through soaking. Many companies use wheat for some varieties of their beer. Then the sugars are added to hops and the two are brewed together. 1  GENETICS Then yeast is added to do its magic and begin the process of fermentation; this can take weeks. After that, the beer is flavored to taste and ready to drink. Agriculture field: The earliest farmers selected and bred the best suited crops, having the highest yields, to produce enough food to support a growing population. It seems to be as early biotechnological application. Waste Management: Biotechnological processes are used for wastewater treatment, gas treatment and disposal of solid wastes in environmental engineering. Also, these processes can be utilized for the production of biogas and hydrogen as new energy resources. Composting plant waste. Sewage treatment. Animal waste. Such technologies improve recycling, facilitating the use of recyclates by producers, enabling better purchasing and sorting decisions by consumers, and improving waste sourcing options for recyclers. Advanced digitalization in waste management and treatment is currently mostly in the innovation phase. 2  GENETICS The recent application for biotechnology: In the Industrial field: Inserting specific genes can be used to: ✓ Improve the efficiency of the waste treatment. ✓ Make consumable goods. ✓ Test soil, air and water for contamination. ✓ Produce food supplements. In the agriculture field: Inserting specific genes can be used to: ✓ Allow plants to be resistant to large applications of pesticides. ✓ Allow plants to tolerate environmental stress such as drought. ✓ Increase the nutritional value of crops. ✓ Allow unacceptable high levels of pesticides to be detected in food crops. 80% of cotton is genetically modified. Much of the soybean, corn, and other common food crops are grow from genetically modified seeds. Cleaning up environmental toxins from the air, soil and water. Bioremediation is a branch of biotechnology that employs the use of living organisms, like microbes and bacteria to decontaminate affected areas. It is used in the removal of contaminants, pollutants, and toxins from soil, water, and other environments. 3  GENETICS In the Medical field: Inserting specific genes can be used to: ✓ Produce drugs such as penicillin and cortisone. ✓ Produce Vaccine. ✓ Make diagnostic lab tests. ✓ Produce human proteins for hemophiliacs and diabetic patients. More than 125 approved drugs are produced using genetic engineering Genetically modified organism (GMO): It is any organism whose genetic material has been altered using genetic engineering techniques. Advantages for preferring micro-organism (MO) over other living organisms: ✓ Grow rapidly in comparison to plants and animals. ✓ Need limited space for growth. ✓ Not affected by animal and plant diseases. ✓ Can be genetically manipulated. ✓ More economic. ✓ Capable of producing a wide variety of enzymes. 4  GENETICS Characteristics of Industrial M O.: ✓ Not harmful to humans, plants, or animals. ✓ Grow rapidly and produce product quickly in large-scale culture. ✓ Grow in simple media and should not preferably require additional growth factors to reduce cost. ✓ Be available in pure culture. ✓ Be genetically stable. ✓ Easily amenable to genetic modification to produce strains with more acceptable properties. ✓ Produce usable substance(s). ✓ Cells are easily separated from the product. ✓ Liable to a suitable and cheaper method of product harvest. Preparation of Industrial M O.: 1- Isolation 2- Enrichment 3- Screening 5  GENETICS 1- Isolation: It refers to obtaining MO that can be achieved through: A- Public Culture Collection. B- Naturally isolated. A- Public Culture Collection: It offers Standard organisms. These offered organism give low yield of the product. There are different collection banks: o American Type Culture Collection (ATCC). o National Collection of Type Cultures (NCTC). o The World Federation for Culture Collections (WFCC). B- Isolated from Nature: Soil is very rich in microbial contents. Isolation methods are based on selection of the desired characteristic. 2- Enrichment: Enrichment culture will increase the number of a given organism relative to numbers of other types in the original inoculums. Enrichment in batch or continuous system on a defined growth media and cultivation conditions are performed to encourage the growth of the organism with desired trait. 6  GENETICS 3- Screening: The pure enriched culture must be screened for the desired property; production of a specific enzyme, inhibitory compound, protein, etc. Selected isolates must also be screened for other important features, such as stability and, where necessary, non-toxicity. Screening for desirable traits (Phenotypically). 7  GENETICS The methods for preserving The Industrial MO: 1- Storage at reduced temperature. 2- Storage in a dehydrated form. 3- Other storage methods. The MO must be preserved properly to: ✓ Reduce chances of occurrence of mutations. ✓ Protect against contamination. ✓ Retain viability. 1- Storage at reduced temperature. A- Storage on Agar Slopes: ✓ Stored at 4 C, -20, or -80C. ✓ Sub cultured at approximately 6-monthly intervals. ✓ If covered with sterile mineral oil could be stored for one year. Advantages: 1- Simple. 2- Cheap. Disadvantages: 1- Short term preservation (must be sub-cultured time intervals). 2- Contaminations and/or mutations may occur during regular sub- culturing. 8  GENETICS B- Storage Under Liquid Nitrogen: It uses the liquid or vapor phase of nitrogen at – 150C to – 196C. It is Used for preserving MO and cultured cells such as fungi, animal cells & tissue cultures. This technique involves growing a culture to the maximum stationary phase then re-suspending the cells in a cryo protective agent (such as 10 % glycerol). The suspension is freezed in sealed ampoules before storage under liquid nitrogen. Advantages: ✓ It is Suitable for all MO &tissue cultures. ✓ It provides long term preservation. Disadvantages: ✓ Nitrogen may be evaporated. ✓ Expensive. ✓ Difficulty of shipment. 9  GENETICS 2- Lyophilization or Freeze Drying: Lyophilization, or freeze-drying, involves the freezing of a culture followed by its drying under vacuum which results in the sublimation of the cell water. It is a useful method for long-term preservation. Advantages: ✓ Thermolabile material can be dried. ✓ Its reconstitution is easy as it is porous & uniform. ✓ Avoidance of denaturation. ✓ Avoidance of salts & other solutes migration. 10  GENETICS ✓ Minimal loss of volatile material. ✓ Moisture level can be kept as low as possible. ✓ Sterility can be maintained. Disadvantages: ✓ Long time process. ✓ According to the required product, it may be costly. ✓ Due to porosity & large surface area, the products may be prone to oxidation. The oxidation can be avoided if the product oxidation, the product packed in vacuum or using inert gas or in a container impervious to gases. 3- Other storage methods: Preservation in distilled water. Preservation under oil. Preservation on beads. Storage over silica gel. 11  GENETICS DNA manipulation DNA (Deoxy Ribonucleic acid) manipulation: It is a process that uses laboratory-based technologies to alter the DNA makeup of an organism. This may involve changing a single base pair (A-T or C-G), deleting a region of DNA or adding a new segment of DNA. DNA (Deoxy Ribonucleic acid): ✓ The nucleotides are arranged in chains linked together by phosphodiester bond; between phosphate on C5 of deoxy ribose of one nucleotide and OH on C3 of the next sugar. Assembly of double strands of DNA: 12  GENETICS DNA manipulation techniques: 1- Cutting DNA molecules. 2- Joining DNA molecules. 3- Cloning. 4- Others… 1- Cutting DNA Molecules: The DNA isolated from any type of cell can be fragmented using restriction endo-(inside) nucleases - (cuts nucleic acid). The restriction enzymes are molecular scissors that cut double stranded DNA molecules at specific points - “recognition sequence”. ✓ They are found naturally in a wide variety of prokaryotes. ✓ They are important tools for manipulating DNA: o Restriction enzymes are most widely used in recombinant DNA technology. o They are used in gene cloning and protein expression experiments. Insulin Production. ✓ The Biological role of restriction enzymes: o Most bacteria use restriction enzymes as a defense against bacteriophages. o Restriction enzymes prevent the replication of the phage by cleaving its DNA at specific sites. o The host DNA is protected by Methylases which add methyl groups to adenine or cytosine bases within the recognition site thereby modifying the site and protecting the DNA. 13  GENETICS ✓ Restriction Enzyme cut DNA at specific points known as ‘restriction sites’ which sometimes are palindromic sequences (complementary sequences with identical nucleotide sequences when read in the direction 5 ̀ to3 ̀. ✓ Endonuclease recognition sequence is usually 4-6 base pairs. ✓ The enzyme "scans" a DNA molecule, looking for a definite sequence. ✓ As, it finds this recognition sequence, it stops and cuts the strands. ✓ On double stranded DNA the recognition sequence is on both strands, but runs in opposite directions. This allows the enzyme to cut both strands. ✓ The restriction enzyme makes one cut in each of the sugar phosphate backbones of the double helix by hydrolyzing the phoshpho-diester bond. Specifically, the bond between the 3’ O atom and the P atom is broken. ✓ 3’OH and 5’ PO4 is produced. ✓ Mg2+ is required for the catalytic activity of the enzyme. Mechanism of Action of Restriction Enzymes 14  GENETICS ✓ EcoRI (pronounced "eco R one") is a restriction endonuclease enzyme isolated from species E. coli. ✓ The Eco part of the enzyme's name originates from the species from which it was isolated, while the R represents the particular strain. ✓ EcoRI has specificity for the sequence GAATTC. ✓ Restriction enzymes, like EcoRI, that generate sticky ends. ✓ EcoRV restriction enzyme generates blunt ends. ✓ BamHI, these enzymes are named by the bacteria and the strain from which they are isolated. For BamHI this is Bacillus amyloliquefaciens strain H. ✓ BamHI has specificity for the sequence GGATCC. 15  GENETICS Sticky ends are also known as overhanging ends. Sticky ends are possessed with unpaired bases and require complementary bases to form bonds. Blunt ends are also known as non-overhanging ends since they do not have 3’ and 5’ overhanging bases at the ends. Both strands terminate from base pairs in blunt ends. Sticky ends or blunt ends can be used to join DNA fragments. Sticky ends are more cohesive compared to blunt ends. 2- Joining of DNA Molecules: If two pieces of DNA have matching ends, they can link together to form a single, unbroken molecule of DNA using DNA ligase enzyme. This is enzyme known as a DNA-joining enzyme. The mechanism of DNA ligase is to form two covalent phosphodiester bonds between: ✓ 3 'hydroxyl ends of one nucleotide "acceptor " and 5 ' phosphate end of another "donor". Energy is required for the ligase reaction. 16  GENETICS Types of DNA ligase: 1- E. coli DNA ligase. 2- T4 DNA ligase. 3- Mammalian DNA ligases. 4- Thermostable DNA ligases. Uses of DNA ligase: ✓ DNA ligase is used in both DNA repair and DNA replication. ✓ DNA ligase has extensive use in molecular biology laboratories for recombinant DNA experiments. ✓ Purified DNA ligase is used in gene cloning to join DNA molecules together to form recombinant DNA. 3- Cloning: DNA cloning is a molecular biology technique that makes many identical copies of a piece of DNA, such as a gene. Genes present in DNA fragments that have been excised with restriction endonucleases can be replicated and expressed, by ligating them into different types of vectors. In gene cloning, once recombinant DNA (rDNA) has been constructed it is introduced into a host. In the host, rDNA has to be: ✓ Maintained. ✓ Replicated. ✓ Passed from one generation to another. 17  GENETICS This is achieved by introducing rDNA into a cell on a DNA vehicle called a cloning vector. Vectors: ✓ These are relatively small DNA molecules that have the ability to replicate readily and independently from the chromosome. ✓ The desired gene (ligated gene carried on the vector) can be inserted to host cell to produce several copy numbers of the desired gene.\ Cloning vectors: 1- Plasmids. 2- Bacteriophages. 3- Cosmid. ✓ Most vectors are genetically engineered plasmids or phages. ✓ There are also bacterial artificial chromosomes (BAC), and yeast artificial chromosomes (YAC). 18  GENETICS Characteristics of cloning vectors: o It must be small in size. o It must be self-replicating inside host cell, o It has an origin of Replication (ORI): a specific sequence of nucleotides from where replication starts. o It must possess restriction site for restriction endonuclease enzymes (multiple cloning site; MCS or poly linker). o The introduction of donor DNA fragment must not interfere with replication property of the vector. o It must possess some marker gene (ex: gene conferring resistance to definite antibiotic). So, it can be used for later identification of recombinant cell. 1- Plasmids: Extra-chromosomal DNA found in bacteria. Loops of double-stranded DNA. Can hold up to 10 kb fragments. Some of them present in multiple copies. It independently replicates inside bacteria using their own origin of replication (ORI). “Autonomous replication”. It has Several MCS. Restriction sites of the polylinker are not present anywhere else in the plasmid. 19  GENETICS Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector. A selectable marker usually an antibiotic resistance gene. 2- Bacteriophages: The most popular bacteriophage is the bacteriophage that attacks E.coli (lambda λ bacteriophage) , which is made a tubular protein tail and a protein head packed with approximately 50 kb of DNA. Out of the 50 kb that make the bacteriophage, less than half are essential for the propagation of the phage and around 15:20 kb can be replaced for recombinant DNA; hence their name replacement vectors. 20  GENETICS ʎ Bacteriophages DNA is linear double stranded DNA with small single- stranded complementary DNA fragments at each end called ‘cos ends’ ‘cohesive end’. Recombinant phages can be packed into phage particles by enzymes which recognize and process the cos ends, provided that they are 35-45 kb apart. The in vitro packaging results in the formation of recombinant phages that can be transduced to E. coli cells. 21  GENETICS 22  GENETICS 3- Cosmid: A cosmid is a type of hybrid plasmid that contains a Lambda phage cos sequence. Cosmids (cos sites + plasmid = cosmids). Cosmids are cloning vectors that can carry up to 40 kb of cloned DNA and can also be maintained in E. coli. 23  GENETICS 24  GENETICS The required characteristics for microbial host for cloning vectors: ✓ Well characterized genetically and biochemically. ✓ Rapid grower in cheap media. ✓ Not pathogenic. ✓ Free from harmful restriction endonucleases activity to avoid degradation of cloned gene. ✓ Defective in homology-based recombination to avoid integration “vector should be autonomous”. Microorganisms most commonly used for cloning are: ✓ E. coli, Bacillus subtilis, and Saccharomyces cerevisiae. ✓ E. coli disadvantages: o Their endotoxin contents. o Its tendency to colonize in human intestine. 25  GENETICS o Its potential pathogenicity. ✓ Bacillus subtilis does not produce endotoxin and it is non- pathogenic. ✓ Its main problems are: o The plasmid and foreign gene instabilities. o The loss of plasmid replication ability upon repeated subculture. Introduction of Vector into Host cell There are four methods for expression and maintenance of recombinant genes: 1- Electroporation. 2- Transformation. 3- Transduction. 4- Conjugation. The Competent cells: Competent cells are cells (E. coli) that have been specially treated to transform efficiently. There are two types of competent cells. ✓ Electrocompetent. 26  GENETICS ✓ Chemically Competent. 1- Electroporation. Electroporation is the artificial means of DNA transfer into the cells using an electrical field. Induction of free DNA uptake by the bacterium after subjecting it to a high electric field. In which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or DNA to be introduced in to the cell (also called electro-transfer). Electroporation allows the uptake of most sizes of plasmids. The cells are placed in an electroporation device that delivers a pulse of electricity to disrupt the membranes of the cells allowing the plasmids to enter the cells. Electrocompetent formats provide the highest introduction efficiencies, but do require an electroporation device. 27  GENETICS 2- Transformation: E. coli can uptake recombinant plasmid DNA by treating the cells with ice-cold CaCl2 until they reach a ‘competent’ state in which they are ready to take up DNA. These cells are then presented with the recombinant plasmid and exposed briefly to a heat shock of 42°C which enables them to take up the DNA. Chemically competent cells are treated with a buffer that contains CaCl2 and other salts that disrupt the cell membrane creating “holes” that allow the plasmids to pass into the cell. Most researchers use chemically competent cells because they are less expensive, can be made in the lab and do not require special equipment. 3- Transduction: The transfer of recombinant non-viral DNA to a cell is achieved by a virus. This is the method of choice for the introduction of recombinant bacteriophages and cosmids into E. coli cells. 28  GENETICS 4- Conjugation: Direct contact through cell–cell junctions. Only plasmid cloning vectors containing conjugative elements can be transferred by conjugation. Conjugative elements: are a diverse group of mobile genetic elements found in both Gram- positive and Gram-negative bacteria. 29  GENETICS Optimizing expression of recombinant genes: The primary objective of pharmaceutical companies involved in the production of recombinant drugs is the maximal expression of recombinant genes to generate large quantities of these drugs. Unfortunately, the cloning of a gene into a vector does not ensure that it will be highly expressed. 30  GENETICS Therefore, to improve expression of a gene we have to optimize the different stages that lead to the synthesis of a protein. This is achieved by the use of so-called expression vectors. Expression vectors: An expression vector is one which contains, in addition to the cloned gene, all regulatory sequences required for expression of the cloned gene. Elements of gene expression are those involved in transcription and translation. Expression vector is constructed, and should have the following properties: ✓ Can achieve high copy number. The higher the copy number, the better expression of the gene. ✓ Has high efficiency promotor site. ✓ The promotor must be well regulated. ✓ Its mRNA must have the proper ribosome binding site. 31  GENETICS Optimizing transcription: To optimize transcription, it must be ensured that the recombinant gene is placed after a promoter that will be recognized by the RNA polymerase of the host cell where the gene is going to be expressed. There are two types of promoters that can be selected: ✓ Constitutive promoters: which are expressed all the time. ✓ Inducible promoters. where expression is turned off during culture growth and turned on upon the addition of an inducible molecule to the culture (ex: lac operon), usually shortly before harvesting, when high numbers of bacteria are present in the culture. Inducible promoters are very useful when expressing genes coding for foreign toxic proteins as their premature expression could lead to growth impairments and consequently low yields of recombinant protein. Furthermore, to ensure that transcription finishes after the 3’-end of the recombinant gene, a transcriptional terminator/ stop must be placed just downstream of this gene. Expression vector also, contain poly adenylation site that protect the transcriped mRNA from degradation. Concerning translation and protein synthesis; expression vector should contain Shin Dalgarno’s sequence that allow the attachment of ribosomes to mRNA. 32  GENETICS 33  GENETICS The Post translational modifications: Over-expressed protein may still need to undergo post-translational modifications before it can be active. Some of these modifications include: ✓ Correct disulphide bond. ✓ Additions to amino acids such as: phosphorylation, acetylation, sulphation, acylation, etc. Unfortunately, the popular E. coli host, where most recombinant proteins are expressed, is unable to carry out some of these modifications. Hence, it is essential to select a suitable host for the expression of the target protein that can carry out the required post-translation modifications that will enable the synthesis of large amounts of a biologically active product. The Main Difference – Cloning Vector vs. Expression Vector: Cloning vector and expression vector are two types of vectors, used in recombinant DNA technology to carry foreign DNA segments into a target cell. Both cloning and expression vectors comprise of the origin of replication, unique restriction sites, and selectable marker gene in their vector sequences. Both cloning and expression vectors are self-replicative due to the presence of an origin of replication. Cloning vectors can be either plasmids, cosmids or bacteriophages, while, expression vector is only Plasmid. The main difference between cloning vector and expression vector is that cloning vector is used to carry foreign DNA segments into a host cell, and obtaining several copies of the inserted DNA whereas expression vector is a type of a cloning vector, which contains suitable expression signals with maximal gene expression and protein synthesis. 34  GENETICS 35  GENETICS 36  GENETICS Screening of cloned genes: Blue –White screening for colony selection: It is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. DNA of interest is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal. X-gal is an organic compound consisting of galactose linked to a substituted indole. X-gal is often used in molecular biology to test for the presence of an enzyme, β- galactosidase. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo dye as a result of enzyme-catalyzed hydrolysis. 37  GENETICS Limitations of blue-white screening: The lacZ gene in the vector may sometimes be non-functional and may not produce β-galactosidase. The resulting colony will not be recombinant but will appear white. Even if a small sequence of foreign DNA may be inserted into MCS and change the reading frame of lacZ gene. This results in false positive white colonies. Small inserts within the reading frame of lacZ may produce ambiguous light blue colonies as β-galactosidase is only partially inactivated. 38  GENETICS Gene detection I- Agarose Gel Electrophoresis Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size (isoelectric focusing agarose electrophoresis is essentially size independent), and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix. Agarose gel is easy to cast, has relatively fewer charged groups, and is particularly suitable for separating DNA of size range most often encountered in laboratories, which accounts for the popularity of its use. The separated DNA may be viewed with stain, most commonly under UV light, and the DNA fragments can be extracted from the gel with relative ease. Agarose gel electrophoresis is routinely used for the analysis of DNA; DNA is negatively charged. When it is placed in an electrical field, DNA will migrate toward the positive pole (anode). An agarose gel is used to slow the movement of DNA and separate by size. 39  GENETICS Speed of DNA migration depends on: ✓ Strength of the electrical field, buffer, density of agarose gel… ✓ Size of the DNA. Small DNA move faster than large DNA. Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight. Visualization of the DNA in the gel: ✓ Before electrophoresis, DNA is added to bromophenol blue in order to be added visible during electrophoresis. ✓ After electrophoresis, DNA is visualized by adding Ethidium bromide which chelate with DNA and seen under UV light (UV transillimenator). 40  GENETICS Spectrophotometric measurements: ✓ An important tool for DNA and RNA quantification. ✓ Wavelength 260 For DNA and RNA. ✓ Sample should be sufficiently diluted. ✓ Reliable reading should be range from 0.1 to 1.0. SDS-polyacrylamide gel electrophoresis SDS-PAGE: Native protein carries different R groups with different charge & folded. So, protein is prepared with little disturbance to the cellular material SDS: Sodium Dodecyl Sulfate is a detergent. Protein coated with a negative charge in proportion to its molecular weight. A sample of protein, often freshly isolated and unpurified, is boiled in the detergent sodium dodecyl sulfate and beta-mercaptoethanol. The mercaptoethanol reduces disulfide bonds. The detergent disrupts secondary and tertiary structure. On the molecular level, proteins are stretched out and coated with the detergent (which has a negative charge) by this treatment. They will then migrate through a gel towards the positive pole at a rate proportional to their linear size Molecular weights with respect to size markers may then be determined Polyacrylamide Gel Creates tunnels in gel for molecules to move. 41  GENETICS 42  GENETICS Uses of PAGE: Separates proteins from each other. Proteins separated by size and Isoelectric point. Determines: ✓ Molecular size of protein. ✓ Quantifies the amount present. ✓ Displays Impurities. ✓ Used in western blot assays by antigen interactions. 43  GENETICS II- Blotting techniques. A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA. The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. Enzyme linked immunosorbent assay (ELISA) technique. 44  GENETICS 1- Applications of Southern hybridization: ✓ Restriction fragment length polymorphism (RFLP) & DNA finger printing (Variable number of tandem repeats) VNTRs ✓ Detection of genes. RFLP: RFLP is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. It is a technique that exploits variations in homologous DNA sequences. Can be used in paternity cases or criminal cases to determine the source of a DNA sample (i.e. it has forensic applications). Can be used determine the disease status of an individual. (e.g. it can be used in the detection of mutations particularly known mutations). Can be used to investigate the source and spread of microorganism in outbreaks. In Forensic Medicine (Criminal and victim identification in cases of rape, thieves and murder). Making a probe: ✓ A probe is a small (25-2000 bp) length of DNA or RNA. ✓ Complementary to the sequence (gene) of interest. ✓ Labeled for subsequent detection procedures. 45  GENETICS 46  GENETICS 2- Northern blotting or northern hybridization: Technique for detecting specific RNAs separated by electrophoresis by hybridization to a labeled DNA probe. Used to determine the transcription of a gene. 3- Western blotting, or immunoblotting: Technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies. Western blots allow the determination of the molecular weight of a protein (antibody) and to measure relative amounts of the protein present in different samples. Western blot analysis can detect one protein in a mixture of any number of proteins while giving you information about the size of the protein. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. This antibody is used as a probe to detect the protein of interest. Western Blot Analysis: Identifies protein through antibody interaction. Run proteins on denatured gel (SDS-PAGE). Transfer (blot) proteins onto membrane. Probe the membrane with primary antibody. Add secondary antibody (this antibody is linked to an enzyme). Substrate is added and color appears. 47  GENETICS Flow chart of western blotting: Western blotting: 48  GENETICS 49  GENETICS 4- Enzyme linked immunosorbent assay (ELISA) technique: It is a biochemical technique used in immunology to detect the presence of an antibody or an antigen in a sample and after protein expression to determine the presence of proteins. Forming insulation to allow nerve conduction and prevent heat loss. Deficiencies or imbalance in lipid metabolism leads to some problems as obesity & atherosclerosis. Also, polyunsaturated fatty acids play an important role in nutrition and health. 50  GENETICS DNA Sequencing It is the process of determining the order of bases adenine (A), thymine (T), cytosine (C), and guanine (G) along a DNA strand. All the information required for the growth and development of an organism is encoded in the DNA. So, DNA sequencing is fundamental to genome analysis and understanding the biological processes in general. Methods of sequencing: 1- Sanger and Gilbert sequencing. 2- Pyrosequencing. 3- Illumina sequencing. 1- Sanger sequencing: Sanger sequencing is based on the use of chain terminators, 2,3 dideoxy nucleoside triphosphate (ddNTPs); the nucleotide here may be A, G, T, C. ddNTP if added to growing DNA strands will stop the replication of DNA as it cannot make phosphodiester bond. 51  GENETICS Requirement for the process: ✓ A DNA template (The DNA fragment that need to know its sequence of nucleotide). ✓ DNA Primer specific and complement with the first nucleotide sequence. ✓ DNA polymerase. ✓ Deoxy nucleoside triphosphate; the 4 building blocks of DNA (dATP, dGTP, d CTP, d TTP). ✓ Small number of fluorescent nucleotides dideoxy nucleoside triphosphates (ddNTPs) with 4 different bases; dd ATP, dd GTP, ddCTP, dd TTP. Steps of Sanger technique: ✓ DNA is fragmented, primers are added. ✓ Fluorescent-labeled dideoxynucleotides are used to generate DNA fragments that terminate at each nucleotide along the template strand. ✓ The DNA is separated by capillary electrophoresis on the basis of size. ✓ From the order of fragments formed, the DNA sequence can be read. ✓ The smallest fragments were terminated earliest, and they come out of the column first, so the order in which different fluorescent tags exit the column is also the sequence of the strand. ✓ The DNA sequence readout is shown on an electropherogram that is generated by a laser scanner. 52  GENETICS 53  GENETICS 54  GENETICS 2- Pyrosequencing: The dNTP incorporation into DNA is detected in the form of light. It’s a luminescent method based on the action of two enzymes to accurately detect nucleic acid sequences during the synthesis. Pyrophosphate is released when a dNTP is added to the end of a nascent strand of DNA. Because dNTPs are sequentially added to the reaction and because the pyrophosphate concentration is continuously monitored, the DNA sequence can be determined. This approach measure pyrophosphate production as each nucleotide is washed through the system in turn over the template DNA affixed to a solid phase. Generation of light release by the ppi make the automation possible for this method. Less complex process with fewer steps than sanger sequencing. 55  GENETICS 3- Illumina sequencing (Sequencing by synthesis): DNA fragments are flanked with adaptors. A flat surface coated with two types of primers, corresponding to the adaptors. Amplification proceeds in cycles, with one end of each bridge tied to the surface. Labeled nucleotide, polymerases. In Illumina sequencing, up to 500,000,000 separate sequencing reactions are run simultaneously on a single slide (the size of a microscope slide) put into a single machine. Each reaction is analyzed separately and the sequences generated from all 500 million DNAs are stored in an attached computer. 56  GENETICS Gene Therapy Genes: Are carried on a chromosome. The basic unit of heredity. Encode how to make a protein. Proteins carry out most of life’s function. When the gene is altered causes dysfunction of a protein. When there is a mutation in the gene, then it will change the codon, which will change which amino acid is called for which will change the conformation of the protein which will change the function of the protein. Genetic disorders result from mutations in the genome. 57  GENETICS Gene Therapy: It is a technique for correcting defective genes that are responsible for disease development. There are four approaches: ✓ A normal gene inserted to compensate for a nonfunctional gene. ✓ An abnormal gene repaired through selective reverse mutation. ✓ Change the regulation of gene pairs. ✓ Gene editing. Gene therapy could be very different for different diseases: ✓ Gene transplantation to patient with gene deletion. ✓ Gene correction to revert specific mutation in the gene of interest. ✓ Gene augmentation to enhance expression of gene of interest. 58  GENETICS I- In vivo gene therapy: 1- The genetic material is transferred directly into the body of the patient. 2- More or less random process; small ability to control; less manipulations. 3- Only available option for tissues that cannot be grown in vitro; or if grown cells cannot be transferred back. Ex of vivo gene therapy: 1. The genetic material is first transferred into the cells grown in vitro. 2. Controlled process; Genetically altered cells are selected and expanded; more manipulations. 3. Cells are then returned back to the patient. 59  GENETICS 60  GENETICS Delivery of the genes into the body: A- Non-viral Options: Direct introduction of therapeutic DNA. Only done with certain tissue and requires a lot of DNA. It is Less effective. ✓ Creation of artificial lipid sphere with aqueous core, liposome ✓ Carries therapeutic DNA through membrane ✓ Chemically linking DNA to molecule that will bind to special cell receptors. ✓ DNA is engulfed by cell membrane 61  GENETICS Liposomes: Liposomes are closed bilayer structures spontaneously formed by hydrated phospholipids that are widely used as efficient delivery systems for drugs or antigens, due to their capability to encapsulate bioactive hydrophilic, amphipathic, and lipophilic molecules into inner water phase or within lipid leaflets. 62  GENETICS DNA delivery of genes by liposomes is: Cheaper than viruses. No immune response. Especially good for in-lung delivery (cystic fibrosis) B- Viruses for gene delivery: A vector delivers the therapeutic gene into a patient’s target cell. The target cells become infected with the viral vector. The vector’s genetic material is inserted into the target cell. Functional proteins are created from the therapeutic gene causing the cell to return to a normal state. 63  GENETICS Viruses: Replicate by inserting their DNA into a host cell Gene therapy can use it to insert genes that encode for a desired protein to create the desired trait. There are Four different types: 1- Adenoviruses: They are double stranded DNA genome that cause respiratory, intestinal, and eye infections in humans. The inserted DNA is not incorporate into genome. Not replicated though. It has to be reinserted when more cells divide. E.g: common cold. 64  GENETICS 2- Adeno-associated Viruses: Adeno-associated Virus- small, single stranded DNA that insert genetic material at a specific point on chromosome 19. It is from parvovirus family- causes no known disease and doesn't trigger patient immune response. It has low information capacity. The gene is always "on" so the protein is always being expressed, possibly even in instances when it isn't needed. Hemophilia treatments, for example, a gene-carrying vector could be injected into a muscle, prompting the muscle cells to produce Factor IX and thus prevent bleeding. Patients have not needed Factor IX injections for more than a year. 3- Retroviruses: The retrovirus goes through reverse transcription using reverse transcriptase and RNA. The double stranded viral genome integrates into the human genome using integrase. Integrase inserts the gene anywhere because it has no specific site. May cause insertional mutagenesis. One gene disrupts another gene’s code (disrupted cell division causes cancer from uncontrolled cell division) Vectors used are derived from the human immunodeficiency virus (HIV) and are being evaluated for safety. 65  GENETICS 4- Herpes Simplex Viruses: Double stranded DNA viruses that infect neurons. Ex. Herpes simplex virus type 1. 66  GENETICS Problems with Gene Therapy: Short Lived ✓ Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene therapy from long time. ✓ Would have to have multiple rounds of therapy. Immune Response ✓ Viral Vectors: Patient could have toxic, immune, inflammatory response also may cause disease once inside. Multigene Disorders: ✓ Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are hard to treat because you need to introduce more than one gene. May induce a tumor if integrated in a tumor suppressor gene because insertional mutagenesis. ✓ One problem with gene therapy is that one does not have control over where the gene will be inserted into the genome. ✓ The location of a gene in the genome is of importance for the degree of expression of the gene and for the regulation of the gene (the so- called "position effect"), and thus the gene regulatory aspects are always uncertain after gene therapy. 67  GENETICS Recent Developments: Genes get into brain using liposomes coated in polymer of polyethylene glycol is potential for treating Parkinson’s disease. RNA interference or gene silencing to treat Huntington’s: ✓ siRNAs used to degrade RNA of particular sequence ✓ Abnormal protein won’t be produced. Create tiny liposomes that can carry therapeutic DNA through pores of nuclear membrane. Sickle cell successfully treated in mice. Genome editing: Genome editing, also called gene editing, is an area of research seeking to modify genes of living organisms to improve our understanding of gene function and develop ways to use it to treat genetic or acquired diseases. 68  GENETICS Genome editing tools have two features: 1- Recognize specific DNA sequences (i.e. specific genes or non-coding elements). 2- Cut DNA (“nuclease”), then a scar is left behind. Genome editing: cleavage repair can either disrupt original sequence or replace it with a new copy. Two strategies for genetic therapy: gene addition and genome editing: 69  GENETICS Gene addition: Feasible with existing technology; clinical trials ongoing. Early trial results appear exciting. Challenges: 1. Will enough of the added gene be made in the cells with the integration? Will enough of the blood stem cells have the added gene? 2. Is the benefit durable? Will the added gene continue to function over days, weeks, months, years, decades? 3. Is the added gene safe? Will its semi-random integration into the genome change the function of other genes in the genome? Gene editing: Promise of permanent repair of the underlying disease-causing mutation. Promise of specific beneficial change at the intended genomic site (e.g. b- globin gene) without impacting remainder of genome. Challenges: 1. Technology is in a relatively early stage and needs to be further developed. 2. Can enough cells be edited to have therapeutic impact? 3. Will the editing be exquisitely specific, or will other regions of the genome aside from the target be affected? 70  GENETICS Potential benefits: Modification would be permanent. Survival advantage of cells (would outcompete unmodified cells). Compared to gene addition, no semi-random insertion of material into the genome, and no need for lifelong expression of foreign sequences. Potential risks: Genome modification at sites other than the intended target. Preparation (“conditioning”) therapy for stem cell transplant (shared risk of both gene addition and genome editing; potentially much less toxic than for “allotransplant” (from related or unrelated donor). Clustered regularly interspaced short palindromic repeats (CRISPR): It is a genome editing technique that: 1- Targets a specific section of DNA. 2- Makes a precise cut/break at the target site It can do one of two things: ✓ Makes a gene nonfunctional. ✓ Replaces one version of a gene with another. 71  GENETICS 72  GENETICS References: An Introduction to Genetic Engineering by Desmond S. T. Nicholl Cambridge University Press ”Third Edition” 2007 Applied Molecular Biotechnology The Next Generation of Genetic Engineering by Muhammad Sarwar Khan, Iqrar Ahmad Khan, Debmalya Barh. CRC Press ,Taylor & Francis Group (2016) Techniques in Genetic Engineering by Işıl Aksan Kurnaz. CRC PressTaylor & Francis Group (2015). Biochemistry (Lippincott Illustrated Reviews Series) by Denise R. Ferrier (Lippincott Williams & Wilkins; 6th Edition, 2013) Medical biochemistry by M.D. Chatterjea and Shinde Rana (Jaypee Brothers Medical Pub; 8th edition, 2011) Clinical Chemistry by W.J. Marshall, Márta Lapsley (8th Edition, 2016). Lehninger Principles of Biochemistry by D.L. Nelson, M.M. Cox (6th edition,2012) Journal of Cellular Biochemistry (https://onlinelibrary.wiley.com/journal/10974644) 73