Molecular Biology - 05 Experimental Manipulations

MOLECULAR BIOLOGY AND DIAGNOSTICS STEPS OF GENETIC RECOMBINATION TECHNOLOGY (OVERVIEW) EXPERIMENTAL MANIPULATION OF MACROMOLECULES I. ISOLATION OF GENETIC MATERIAL OUTLINE o Gel Electrophoresis • Recombinant DNA o DNA Microarrays o Recombinant DNA Technology o Steps of Genetic • Applications and Use of Recombination Technology Genetic Engineering/ (Overview) Recombinant DNA Technology • Restriction Enzymes Function o Application of Recombinant as for Pinking Shea DNA Technology o Restriction Enzymes o Process of Developing o Factors Affecting the Activity of Genetically Modified Plant Restriction Enzymes Species o Products of Restriction o Genetically Modified Food Digestion o Medicinal Applications o Applications o Recombinant Vaccines • Genetic Interlopers: Vectors o Genetic Engineering in Gene Function as Vehicle for Therapy Transferring Genes o Limitations of Recombinant o Vectors for Genomic Cloning DNA Technology and Sequencing • Selection of the Genetically o Types of Vectors Modified Organism (GMO) • Detection of Recombinant DNA o Genetically Modified o Methods for Detection of Organism (GMO) Recombinant DNA Ways in Selection of GMO o Polymerase Chain Reaction o Gene Sequence Analysis o Complimentary and Hybridization • • • II. RESTRICTION ENZYME DIGESTION • • • • L1: RECOMBINANT DNA RECOMBINANT DNA TECHNOLOGY • • • • Recombinant DNA technology refers to the joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. Recombinant DNA (rDNA), on the other hand, is the general name for a piece of DNA that has been created by the combination of at least two strands. They are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in genome. Recombinant DNA in a living organism was first achieved in 1973 by Herbert Boyer, of the University of California at San Francisco, and Stanley Cohen, at Stanford University, who used E. coli restriction enzymes to insert foreign DNA into plasmids. The first step in rDNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules. Since DNA exists within the cell membrane along with other macromolecules such as RNA, polysaccharides, proteins, and lipids, it must be separated and purified which involves enzymes such as lysozymes, cellulase, chitinase, ribonuclease, proteases etc. Other macromolecules are removable with other enzymes or treatments. Ultimately, the addition of ethanol causes the DNA to precipitate out as fine threads. This is then spooled out to give purified DNA. • Restriction enzymes act as molecular scissors that cut DNA at specific locations. These reactions are called ‘restriction enzyme digestions’. They involve incubation of purified DNA with selected restriction enzyme, at conditions optimal for that specific enzyme. The technique ‘Agarose Gel Electrophoresis’ reveals the progress of the restriction enzyme digestion. This technique involves running out the DNA on an agarose gel. On the application of current, the negatively charged DNA travels to the positive electrode and is separated out based on size. This allows separating and cutting out the digested DNA fragments. Vector DNA is also processed using the same procedure. III. AMPLIFICATION USING PCR • • • • Polymerase Chain Reaction or PCR is a method of making multiple copies of a DNA sequence using the enzyme – DNA polymerase in vitro. It helps to amplify a single copy or a few copies of DNA into thousands to millions of copies. PCR reactions are run on ‘thermal cyclers’ using the following components: 1. Template – DNA to be amplified 2. Primers – small, chemically synthesized oligonucleotides that are complementary to a region of the DNA. 3. Enzyme – DNA polymerase 4. Nucleotides – needed to extend primers by the enzyme. The cut fragments of DNA can be amplified using PCR and then ligated with the cut vector. IV. LIGATION OF DNA MOLECULES • • • • The purified DNA and the vector of interest are cut with the same restriction enzyme. This gives us the cut fragment of DNA and the cut vector, that is now open. The process of joining these two pieces together using the enzyme ‘DNA ligase’ is ‘ligation’. The resulting DNA molecule is a hybrid of two DNA molecules – the interest molecule and the vector. In the terminology of genetics this intermixing of different DNA strands is called recombination. • Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and the technology is referred to as the recom-binant DNA technology. • V. INSERTION OF RECOMBINANT DNA INTO HOST • • In this step, the recombinant DNA is introduced into a recipient host cell mostly, a bacterial cell. This process is ‘Transformation’. Bacterial cells do not accept foreign DNA easily. Therefore, they are treated to make them ‘competent’ to accept new DNA. The processes used may be thermal shock, Ca++ ion treatment, electroporation, etc. VI. ISOLATION OF RECOMBINANT CELLS • • • • The transformation process generates a mixed population of transformed and non-trans- formed host cells. The selection process involves filtering the transformed host cells only. For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector is employed. For examples, PBR322 plasmid vector contains different marker gene (Ampicillin resistant gene and Tetracycline resistant gene. When pst1 RE is used it knock out Ampicillin resistant gene from the plasmid, so that the recombinant cell become sensitive to Ampicillin. (sticky ends) while others cut without generating such overhangs, instead of producing blunt ends. Molecules with complementary sticky ends can easily anneal, or form hydrogen bonds between complementary bases, at their sticky ends. The annealing step allows hybridization of the single-stranded overhangs. Hybridization refers to the joining together of two complementary single strands of DNA. Blunt ends can also attach together, but less efficiently than sticky ends due to the lack of complementary overhangs facilitating the process. In either case, ligation by DNA ligase can then rejoin the two sugar-phosphate backbones of the DNA through covalent bonding, making the molecule a continuous double strand. In 1972, Paul Berg, a Stanford biochemist, was the first to produce a recombinant DNA molecule using this technique, combining the SV40 monkey virus with E. coli bacteriophage lambda to create a hybrid. L2: RESTRICTION ENZYMES FUNCTION AS FOR PINKING SHEAR RESTRICTION ENZYMES • • • • A protein produced by bacteria or viruses that cleaves DNA at specific sites along the molecule. Bacterial enzymes produced as a protection mechanism to cut and destroy foreign cytoplasmic DNA that is most commonly a result of bacteriophage infection. In recombinant DNA technology, DNA molecules are manipulated using naturally occurring enzymes derived mainly from bacteria and viruses. The creation of recombinant DNA molecules is possible due to the use of naturally occurring restriction endonucleases (restriction enzymes), bacterial enzymes produced as a protection mechanism to cut and destroy foreign cytoplasmic DNA that is most commonly a result of bacteriophage infection. Stewart Linn and Werner Arber discovered restriction enzymes in their 1960s studies of how E. coli limits bacteriophage replication on the infection. Today, we use restriction enzymes extensively for cutting DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules. Each restriction enzyme cuts DNA at a characteristic recognition site, a specific, usually palindromic, DNA sequence typically between four to six base pairs in length. A palindrome is a sequence of letters that reads the same forward as backward. (The word “level” is an example of a palindrome.) Palindromic DNA sequences contain the same base sequences in the 5ʹ to 3ʹ direction on one strand as in the 5ʹ to 3ʹ direction on the complementary strand. A restriction enzyme recognizes the DNA palindrome and cuts each backbone at identical positions in the palindrome. Some restriction enzymes cut to produce molecules that have complementary overhangs TYPES OF RESTRICTION ENZYMES • Based on the composition, characteristics of the cleavage site, and the cofactor requirements, the restriction endonucleases are classified into four groups, Type I, II, III, and IV. Example Types Co-factor Cleavage Enzymes Type I ATP, Cleave at sites away from EcoB, AdoMet, Mg2+ the recognition site; EcoK Possess both restriction and methylase activities Type II Mg2+ Cleave within or at short EcoR I, specific distances from BamH I the recognition Type III ATP, Mg2+ Cleave at sites 25-27 bp EcoP I, from the recognition site Hinf III Type IV Mg2+ Cleave close to or within the recognition sequence; Target modified DNA, such as methylated, hydroxymethylated and glucosylhydroxymethylated DNA • The type II enzymes do not require ATP for their activity and they cleave close to or within the recognition site. These enzymes are the most extensively used enzymes for gene analysis and cloning work, and are classified into several subtypes. Subtype Type II Type IIS Type IIE Type IIF Type IIT Example Enzymes Cleavage within or adjacent EcoR I to the recognition site; Palindromic recognition site; Homodimeric enzyme Cleavage at a defined Fok I distance from the recognition site; Asymmetric recognition site Interact with two recognition Nae I sites Characteristic Features PRODUCTS OF RESTRICTION DIGESTION • • Recognition Sequence • Restriction digestion of double-stranded DNA produces two kinds of ends: Sticky ends and Blunt ends. Blunt ends possess a 5’-phosphate group that promotes ligation. They are universally compatible with other bluntended DNA. o Blunt ends generated by EcoR V Sticky ends are small stretches of single-stranded DNA capable of self-ligation or ligation with a complementary region from another DNA molecule. The sticky ends possess 3’- or 5’-overhangs of 1–4 nucleotides. o 5’ Cohesive end generated by Bln I o 3’ Cohesive end generated by Kpn I Interact with two recognition NgoM IV sites; Both are required for cleavage; Heterotetramic enzyme Contain different subunits Bpu10 I with restriction and modification activity APPLICATIONS Type IIG Comprised of a single polypeptide chain with restriction and modification activity Type II B Cleave both sides of the recognition site Type IIM Eco571 • Bog I Recognize methylated sites DNAI-RO • FACTORS AFFECTING THE ACTIVITY OF RESTRICTION ENZYMES • • • • Depending on the substrate DNA and the reaction conditions, restriction enzymes show a wide variation of cleavage and possible star activity. In order to obtain the desired cleavage, it becomes important to control the following factors: Star activity: Under sub-optimal reaction conditions, some restriction enzymes cleave base sequences at sites different from the defined recognition sequence. In other words, they cleave at non-specific sites. This phenomenon is called star activity. Some of the factors that induce star activity are high salt and glycerol concentration, presence of impurities, excessive enzyme compared to substrate DNA, increased incubation time, or incompatible buffer and cofactor. Methylated DNA: Several DNA molecules are methylated at the recognition site, making them resistant to cleavage by certain restriction enzymes. For example, most E. coli strains express Dam or Dcm methyltransferases that methylate specific recognition sites to form G6mATC and C5mCA/TGG, respectively. G6mATC is resistant to cleavage by Mbo I. Temperature: Most endonucleases optimally digest the target DNA at 37 °C. However, there are some exceptions with lower or higher optimal temperatures. • • • The ability of restriction endonucleases to cleave DNA at specific recognition sites has enabled extensive use of these enzymes as essential tools in several molecular biology techniques. Some of the major applications are explained below: Molecular cloning: A popular application of restriction enzymes has been in the generation of recombinant DNA molecules. The process involves the cutting of the donor DNA (usually a plasmid) and the vector DNA (usually a gene from another organism) by a restriction enzyme to yield compatible ends. These ends could be either ‘blunt’ or ‘sticky’. The two cleaved DNAs are joined together using an enzyme called DNA ligase to generate a recombinant DNA molecule. This recombinant DNA can, then, be introduced into a host organism for replication. DNA mapping, also known as restriction mapping, involves the use of restriction endonucleases to obtain structural information of the DNA fragment or genome. Mapping involves determination of the order of the restriction enzyme sites in the genome. The DNA of interest, whose structure is to be determined, is cleaved with a series of restriction endonucleases to produce DNA fragments varying in size. These fragments are separated on an agarose gel to determine the structure of the DNA of interest. Based on the known restriction enzyme sites of a specific DNA fragment, restriction endonucleases can be used to verify the identity of that DNA fragment. Restriction landmark genomic scanning is a genome analysis method that utilizes a combination of restriction enzymes to visualize differences in methylation levels across the genome of a given organism. It is a useful technique to identify deviations from normal in any DNA. It is very effective in detecting hyper/hypomethylation in tumors, deletions or amplifications of genes, or changes in gene expression throughout the development of an organism. Gene sequencing: A large DNA molecule can be sequenced by digesting it with restriction enzymes and processing resulting fragments through a DNA sequencer. • • • • Restriction fragment length polymorphism (RFLP) involves the digestion of a DNA sample using restriction enzymes, separating these fragments based on length by gel electrophoresis and transferring them onto a membrane. These fragments are then bound to a radioactive or fluorescent labeled probe targeting specific sequences that are bracketed by restriction enzyme sites. An RFLP occurs when the resulting fragment lengths vary between individuals. Each individual has a unique pattern called the ‘biological bar code’. This technique was the first DNA profiling technique used in gene mapping, localization of genes for genetic disorders, determination of risk for disease, and paternity testing. Pulse field gel electrophoresis involves the separation of large DNA fragments, mainly the fragments resulting from digesting a bacterial genome with a rare-cutting restriction enzyme. The unique pattern produced is used to distinguish different strains of bacteria. It may be useful to identify a particular strain as the cause of a widespread disease. Serial analysis of gene expression (SAGE) is a technique that involves the quantitative and simultaneous analysis of a large number of transcripts in the form of small tags. Restriction enzymes are used as an anchoring enzyme and a tagging enzyme in this technique. Restriction enzyme-mediated integration (REMI) involves the use of restriction enzymes to produce compatible cohesive ends in the genome for the insertion of a mixture of plasmid DNA that has been linearized with a restriction enzyme. The plasmid DNA is transformed into the host cell along with a restriction enzyme that facilitates the integration of the DNA into cognate restriction sites in the chromosomes. This technique is useful for genetic screens and for the insertion of genetic and molecular markers at particular points in the genome to identify interesting genes based on their mutant phenotypes. TYPES OF VECTORS PLASMIDS • • • • A small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. Plasmids naturally exist in bacterial cells, and also occur in some eukaryote In recombinant DNA technology, plasmids are often used as vectors, DNA molecules that carry DNA fragments from one organism to another. Plasmids used as vectors can be genetically engineered by researchers and scientific supply companies to have specialized properties. Some plasmid vectors contain genes that confer antibiotic resistance; these resistance genes allow researchers to easily find plasmid-containing colonies by plating them on media containing corresponding antibiotic. Antibiotic kills all host cells that do not harbor the desired plasmid vector, but those that contain vector are able to survive & grow. Plasmids are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell. Plasmid vectors minimalistically consist of an origin of replication that allows for semi-independent replication of the plasmid in the host and also the transgene insert. Plasmid vectors used for cloning typically have a polylinker site, or multiple cloning site (MCS). L3: GENETIC INTERLOPERS: VECTORS FUNCTION AS VEHICLE FOR TRANSFERRING GENES VECTORS FOR GENOMIC CLONING AND SEQUENCING • • • • • In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker. The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as “backbone” of the vector. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express insert in target cell. Expression vectors (expression constructs) are specifically for the expression of the transgene in the target cell, and generally have a promoter sequence that drives expression of the transgene. Transcription vectors are only capable of being transcribed but not translated: they can be replicated in a target cell but not expressed, unlike expression vectors. Transcription vectors are used to amplify their insert. Insertion of a vector into the target cell is usually called transformation for bacterial cells, and transfection for eukaryotic cells, although the insertion of a viral vector is often called transduction. VIRAL VECTORS • Viral vectors are generally genetically-engineered viruses carrying modified viral DNA or RNA that has been rendered noninfectious, but still contain viral promoters and also the transgene. This allows for translation of transgene through a viral promoter. Because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Viral vectors are often designed for permanent incorporation of insert into the host genome, and thus leave distinct genetic markers in the host genome after incorporating the transgene. Retroviruses leave a characteristic retroviral integration pattern after insertion that is detectable and indicates that viral vector has incorporated into host genome. ARTIFICIAL CHROMOSOME VECTORS • • • COSMIDS • 1. 2. 3. • • Cosmids are plasmid vectors that contain cos sites. The cos site is the only requirement for DNA to be packaged into a phage particle. Cosmids were developed in light of this observation. How do you clone into cosmid vectors? Clone the DNA into vector as you would with any plasmid. Introduce the DNA into bacterial cell via a phage particle. Propagate as plasmid. Since phage particles can accept between 38 and 53 kb of DNA and since most cosmids are about 5 kb, between 33 and 48 kb of DNA can cloned in these vectors. Problems associated with lambda and cosmid cloning. 1. Since repeats occur in eukaryotic DNA rearrangements can occur via recombination of repeats present on DNA inserted into lambda or cosmid. 2. Cosmids are difficult to maintain in a bacterial cell because they are somewhat unstable. • It was developed by Professor Mitsuo Oshimura et al. in Tottori University Graduate School of Medicine by deleting gene regions unnecessary for the maintenance and division of chromosomes in the cell from human or mouse chromosomes. They are called HAC (human artificial chromosomes: artificial chromosomes derived from human chromosomes) vector or MAC (mouse artificial chromosomes: artificial chromosomes derived from mouse chromosomes) vector. Since these artificial chromosome vectors inherit the properties of chromosomes, they have quite different properties from conventional plasmid/viral vectors. In the method of constructing a stable gene expression cell line from a plasmid/viral vector or the like, it was necessary to insert the vector into which the desired foreign gene was integrated on the host chromosome in order to maintain it. During this, the vector was randomly inserted on the host chromosome, and the copy number was also variable, which made it difficult to control. Artificial chromosome vectors have the following advantages compared to conventional vectors. Use for various applications is expected, such as the generation of a stable gene expression cell line, preparation of a transgenic animal, and construction of high protein producing cells. L4: DETECTION OF RECOMBINANT DNA METHODS FOR DETECTION OF RECOMBINANT DNA I. II. III. IV. V. PCR (Polymerase Chain Reaction) Gene Sequence Analysis Complimentary and Hybridization Gel Electrophoresis DNA Microarrays • POLYMERASE CHAIN REACTION (PCR) • • Sometimes called "molecular photocopying," the polymerase chain reaction (PCR) is a fast and inexpensive technique used to "amplify" - copy - small segments of DNA. Because significant amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification. Often heralded as one of the most important scientific advances in molecular biology, PCR revolutionized the study of DNA to such an extent that its creator, Kary B. Mullis, was awarded the Nobel Prize for Chemistry in 1993. GENE SEQUENCE ANALYSIS • • • • WHAT IS THE USE OF PCR? • • Once amplified, DNA produced by PCR can be used in many different laboratory procedures. Most mapping techniques in Human Genome Project (HGP) relied on PCR. PCR is also valuable in a number of laboratory and clinical techniques, including DNA fingerprinting, detection of bacteria or viruses (particularly AIDS), and diagnosis of genetic disorders. HOW DOES PCR WORK? • To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures, or separates into two pieces of single-stranded DNA. Next, an enzyme called "Taq polymerase" synthesizes - builds - two new strands of DNA, using the original strands as templates. This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA. Then each of these strands can be used to create two new copies, and so on, and so on. The cycle of denaturing and synthesizing new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment. The entire cycling process of PCR is automated and can be completed in just a few hours. It is directed by a machine called a thermocycler, which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis. The sequence of DNA base pairs can be analyzed by: Restriction mapping - Construct a "road map" of restriction sites. DNA Sequence Analysis - Cut and clone various restriction fragments, and determine the exact sequence of base pairs. All sequence information is deposited in GenBank. If you just want the sequence of the peptide translated from the RNA, you have to look for insulin mRNA or cDNA. Once we have a piece of DNA cloned, it is amplified (available in many copies) and we now have a living clone which provides, in theory, an indefinite source of the DNA sequence. HOW DO WE ANALYZE THE SEQUENCE? • DNA Sequence Determination o Once you have identified a particular region of DNA of interest, you need to find out the precise sequence of DNA nucleotides. This is done by di-deoxy sequencing, in which a DNA polymerase is put together with dNTPs in four different reactions, each containing a small amount of one di-deoxy NTP (ATP, TTP, CTP, or GTP). The di-deoxy nucleotide lacks a 3'OH to continue chain extension, so the chain terminates. Each reaction produces a population of fragments terminated at A, T, C, or G. o The fragments are either radiolabeled or enzymatically labeled. They can be separated on a gel, or on a fluorescence analyzer. All published DNA sequences in the world are deposited in GenBank. COMPLIMENTARY AND HYBRIDIZATION HOW DO WE FIND GENES OF INTEREST IN A GENOME? OR A PARTICULAR MRNA IN THE TOTAL CELLULAR RNA? OR A PARTICULAR PROTEIN OUT OF ALL CELL PROTEIN? • • • • • • In solution, hybrid molecular complexes (usually called hybrids) of the following types can exist (other combinations are possible): DNA-DNA. A single-stranded DNA molecule (ssDNA probe) can form a double-stranded, base-paired hybrid with a ssDNA target if the probe sequence is the reverse complement of the target sequence. A radiolabeled DNA probe can be applied to DNA from a gel transferred to a membrane, called a Southern Blot (named for its inventor). DNA-RNA. A single-stranded DNA (ssDNA) probe molecule can form a double-stranded, base-paired hybrid with an RNA (RNA is usually a single-strand) target if the probe sequence is the reverse complement of the target sequence. An RNA can be radiolabeled to probe a Southern Blot; or, a ssDNA probe can be applied to membrane-bound RNA, called a Northern Blot (name is a pun on Southern.) Protein-Protein. An antibody probe molecule (antibodies are proteins) can form a complex with a target protein molecule if the antibody's antigen-binding site can bind to an epitope (small antigenic region) on the target protein. In this case, the hybrid is called an 'antigen-antibody complex' or 'complex' for short. A radiolabeled antibody can probe membrane-bound proteins, called a Western Blot (an even worse pun.) There are two important features of hybridization: o Hybridization reactions are specific - the probes will only bind to targets with complimentary sequence (or, in the case of antibodies, sites with correct 3-d shape). o Hybridization reactions will occur in the presence of large quantities of molecules similar but not identical to the target. That is, a probe can find one molecule of target in a mixture of zillions of related but noncomplementary molecules. These properties allow you to use hybridization to perform a molecular search for one DNA molecule, or one RNA molecule, or one protein molecule in a complex mixture containing many similar molecules. o • Western Protein denatured with SDS Antibody against protein X, labeled with radio activity or enzyme What do you learn Restriction How much How much from it? map of gene gene X protein X is X in mRNA is present? How chromosome present? How big is protein long is gene X X? mRNA? What is separated by molecular weight? (target) Probe GEL ELECTROPHORESIS SOUTHERN, NORTHERN, AND WESTERN BLOTS • • Blots are named for the target molecule. o Southern Blot-- DNA cut with restriction enzymes probed with radioactive DNA. o Northern Blot--RNA - probed w/ radioactive DNA or RNA. ▪ Example--used to measure angiopoietin angiopoietin expression from cDNA in a transgenic mouse. o Western Blot--Protein - probed with radioactive or enzymatically-tagged antibodies. These molecules must then be immobilized on a solid support, so that they will remain in position during probing and washing. The probe is then added, the nonspecifically bound probe is removed, and the probe is detected. The place where the probe is detected corresponds to the location of the immobilized target molecule. This process is diagrammed below: In the case of Southern, Northern, and Western blots, the initial separation of molecules is done on the basis of molecular weight, by gel electrophoresis. o Preparing for Blots: ▪ Southern Blots - DNA is first cut with restriction enzymes and the resulting double-stranded DNA fragments have an extended rod conformation without pre-treatment. ▪ Northern Blots - Although RNA is singlestranded, RNA molecules often have small regions that can form base-paired secondary structures. To prevent this, the RNA is pretreated with formaldehyde. ▪ Western Blots - Proteins have extensive 2' and 3' structures and are not always negatively charged. Proteins are treated with the detergent SDS (sodium dodecyl sulfate) which removes 2' and 3' structure and coats the protein with negative charges. Transfer to Solid Support. After the DNA, RNA, or protein has been separated by molecular weight, it must be transferred to a solid support before hybridization. (Hybridization does not work well in a gel.) This transfer process is called blotting and is why these hybridization techniques are called blots. Usually, the solid support is a sheet of nitrocellulose paper (sometimes called a filter because the sheets of nitrocellulose were originally used as filter paper), although other materials are sometimes used. DNA, RNA, and protein stick well to nitrocellulose in a sequence-independent manner. o After a series of treatment steps, the probe is added. The probe hybridized to the target molecules is visualized either by autoradiography or by enzyme reaction. Southern DNA cut with restriction enzymes Radioactive gene X DNA Northern RNA denatured w/ formaldehyde Radioactive gene X DNA DNA MICROARRAYS WHAT IS A DNA MICROARRAY? • • Scientists know that a mutation - or alteration - in a particular gene's DNA may contribute to a certain disease. However, it can be very difficult to develop a test to detect these mutations, because most large genes have many regions where mutations can occur. For example, researchers believe that mutations in the genes BRCA1 and BRCA2 cause as many as 60 percent of all cases of hereditary breast and ovarian cancers. But there is not one specific mutation responsible for all of these cases. Researchers have already discovered over 800 different mutations in BRCA1 alone.The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes like BRCA1 and BRCA2. The chip consists of a small glass plate encased in plastic. Some companies manufacture microarrays using methods similar to those used to make computer microchips. On the surface, each chip contains thousands of short, synthetic, singlestranded DNA sequences, which together add up to the normal gene in question, and to variants (mutations) of that gene that have been found in the human population. • • • The researcher then denatures the DNA in the samples - a process that separates the two complementary strands of DNA into single-stranded molecules. The next step is to cut the long strands of DNA into smaller, more manageable fragments and then to label each fragment by attaching a fluorescent dye (there are other ways to do this, but this is one common method). The individual's DNA is labeled with green dye and the control - or normal DNA is labeled with red dye. Both sets of labeled DNA are then inserted into the chip and allowed to hybridize - or bind - to the synthetic DNA on the chip. If the individual does not have a mutation for the gene, both the red and green samples will bind to the sequences on the chip that represent the sequence without the mutation (the "normal" sequence). If the individual does possess a mutation, the individual's DNA will not bind properly to the DNA sequences on the chip that represent the "normal" sequence but instead will bind to the sequence on the chip that represents the mutated DNA. WHAT IS A DNA MICROARRAY USED FOR? • • • When they were first introduced, DNA microarrays were used only as a research tool. Scientists continue today to conduct large-scale population studies - for example, to determine how often individuals with a particular mutation actually develop breast cancer, or to identify the changes in gene sequences that are most often associated with particular diseases. This has become possible because, just as is the case for computer chips, very large numbers of 'features' can be put on microarray chips, representing a very large portion of the human genome. Microarrays can also be used to study the extent to which certain genes are turned on or off in cells and tissues. In this case, instead of isolating DNA from the samples, RNA (which is a transcript of the DNA) is isolated and measured. Today, DNA microarrays are used in clinical diagnostic tests for some diseases. Sometimes they are also used to determine which drugs might be best prescribed for particular individuals, because genes determine how our bodies handle the chemistry related to those drugs.With the advent of new DNA sequencing technologies, some of the tests for which microarrays were used in the past now use DNA sequencing instead. But microarray tests still tend to be less expensive than sequencing, so they may be used for very large studies, as well as for some clinical tests. HOW DOES A DNA MICROARRAY WORK? • To determine whether an individual possesses a mutation for a particular disease, a scientist first obtains a sample of DNA from the patient's blood as well as a control sample one that does not contain a mutation in gene of interest. L5: APPLICATIONS AND USE OF GENETIC ENGINEERING/ RECOMBINANT DNA TECHNOLOGY APPLICATION OF RECOMBINANT DNA TECHNOLOGY • • • • Genetic engineering has great industrial and agricultural value. It is practiced in medicine, genetic research, agriculture, crop improvement, and for production of therapeutic drugs. It is also used in the development of genetically modified organisms. Here we are discussing some of the important applications of genetic engineering. The recombinant DNA technology is used in the crop improvement and development of new economically important traits. Some of them are: o Herbicide resistance o Virus resistance o Delayed fruit ripening o Altered oil content o Pollen control o Development of cold and drought-tolerant plant species. A classical example of it is the BT cotton- one of the types of genetically modified species provides resistance to the plant against bacillus thuringiensis. PROCESS OF DEVELOPING GENETICALLY MODIFIED PLANT SPECIES • • A gene of interest is isolated from the organism using restriction digestion or amplified by the polymerase chain reaction. Recombinant DNA is constructed by inserting a gene of interest into the plasmid, here the T- plasmid is used. In the next step, the T- plasmid is inserted into the agrobacterium. In the last step, the plant species is infected with the transformed bacterial cells and cultured. The entire process of it is shown in the figure below, MEDICINAL APPLICATIONS • • • • • Low-cost drugs, hormones, enzymes, and vaccines are created using genetic engineering tools. The anti-blood-clotting factor is the best example of it in which the plasminogen activating enzyme which is capable of dissolving the blood clot is artificially designed and used in the patients with coronary artery disease or heart attack. Other examples are two other therapeutic proteins somatostatin and lymphokines which are worked against several disease conditions and can be synthesized artificially. Insulin is yet a classic example of a therapeutic protein designed using genetic engineering technology. A gene for insulin is isolated by restriction digestion or through PCR and inserted int the plasmid. The recombinant plasmid DNA is immediately inserted into the bacterial or yeast cell in which the plasmid is multiplying. As the microorganism starts dividing it starts making artificial insulin. A large amount of insulin produced using the same technique at an industrial scale. The detailed outline of insulin production is shown in the figure below, GENETICALLY MODIFIED FOOD • • • • • • • • GMF (genetically modified food) is another best application of genetic engineering in which economically important food products are constructed using recombinant DNA technology. The classical example of it is Flavr Savr tomato, a genetically modified tomato species made up of the antisense RNA technology. It has great economic values as the GM- tomato can easily be transported from one place to another place. Another important application of genetic engineering is genetically modified or genetically engineered food. The quality of some of the food products such as cotton, corn, and soybeans are improved using the present recombinant DNA technology. The aim of developing genetically modified crops or plant species is to make them economical important, nutritious, protein-rich, disease, and stress resistance. Even, using genetic engineering and tissue culture techniques insecticides resistance plant species in tobacco, potato, corn, and cotton are developed. In addition to this, some modified plants capable of generating their own fertilizers can also be created using the present genetic modification technique. Transgenic model organisms are developed to test different parameters- the function of certain genes can be determined by designing the transgenic microorganism and animal models. Harmful pathogens and insecticidal pasts can be destroyed using genetically modified microorganisms which are capable of degrading toxics. • The commercial production of insulin started after the FDA approval in 1982. RECOMBINANT VACCINES • • Vaccines against smallpox, herpes simplex virus and hepatitis are produced using the genetic engineering technique. The vaccines are the inactivated viral particles used to induce an immune response against that pathogen, however, the chance of contamination is high in it. Using the recombinant DNA technology scientists has created a unique type of vaccines that only contains the DNA for viral coat protein thus the pathogen can never be activated again. The main advantage of it is that it is safer, contamination-free and more reactive. GENETIC ENGINEERING IN GENE THERAPY • • Using the gene therapy or gene transfer technique, inherited genetic disorders can be cured. Cystic fibrosis, Duchenne muscular dystrophy and sickle cell anemia like gene therapies are now under the final clinical trial phase and ready to use on patients. In the gene therapy, a faulty, non-function, or mutated gene is replaced with the wild type one using the same technique as explained above. LIMITATIONS OF RECOMBINANT DNA TECHNOLOGY • • • • • • • • • Destruction of native species in the environment the genetically modified species are introduced in. Resilient plants can theoretically give rise to resilient weeds which can be difficult to control. Cross contamination and migration of proprietary DNA between organisms. Recombinant organisms contaminating the natural environment. The recombinant organisms are population of clones, vulnerable in exact same ways. A single disease or pest can wipe out the entire population quickly. Creation of superbug is hypothesized. Ethical concern about humans trying to play God and mess with the nature’s way of selection. It is exaggerated by the fear of unknown of what all can be created using the technology and how is it going to impact the civilization. Such a system might lead to people having their genetic information stolen and used without permission. Many people worry about the safety of modifying food and medicines using recombinant DNA technology. L6: SELECTION OF THE GENETICALLY MODIFIED ORGANISM (GMO) GENETICALLY MODIFIED ORGANISM (GMO) • • • is an organism whose genome has been engineered in the laboratory in order to favour the expression of desired physiological traits or the generation of desired biological products. In conventional livestock production, crop farming, and even pet breeding, it has long been the practice to breed select individuals of a species in order to produce offspring that have desirable traits. In genetic modification, however, recombinant genetic technologies are employed to produce organisms whose genomes have been precisely altered at the molecular level, usually by the inclusion of genes from unrelated species of organisms that code for traits that would not be obtained easily through conventional selective breeding. Genetically modified organisms (GMOs) are produced using scientific methods that include recombinant DNA technology and reproductive cloning. In reproductive cloning, a nucleus is extracted from a cell of the individual to be cloned and is inserted into the enucleated cytoplasm of a host egg (an enucleated egg is an egg cell that has had its own nucleus removed). The process results in the generation of an offspring that is genetically identical to the donor individual. The first animal produced by means of this cloning technique with a nucleus from an adult donor cell (as opposed to a donor embryo) was a sheep named Dolly, born in 1996. Since then a number of other animals, including pigs, horses and dogs, have been generated by reproductive cloning technology. Recombinant DNA technology, on the other hand, involves the insertion of one or more individual genes from an organism of one species into the DNA (deoxyribonucleic acid) of another. Whole-genome replacement, involving the transplantation of one bacterialgenome into the “cell body,” or cytoplasm, of another microorganism, has been reported, although this technology is still limited to basic scientific applications. WAYS IN SELECTION OF GMO I. BLUE-WHITE SCREENING • • to distinguish nonrecombinant from recombinant transformants. lac selection, (also called, "alpha-complementation") II. ANTIBIOTIC RESISTANCE MARKER IV. RESTRICTION DIGESTION • 1. 2. 3. 4. • • In the case of pUC18, the vector carries a selectable marker gene for resistance to the antibiotic ampicillin. The ampicillin resistance genes carried by the recombinant plasmids produce an enzyme, B-lactamase, that is essential to its antibiotic action. If the plasmid vector is introduced into a plasmid-free antibiotic-sensitive bacterial cell, the cell becomes resistant to ampicillin. Extraction of Plasmid Treatment with restriction enzyme Electrophoresis digests on agarose gel Comparison of the sizes of the restriction digest products with those that are expected, based on a restriction map of the desired clone. V. DETECTING PROTEINS EXPRESSED IN THE CLONING HOST • Use of antibodies to detect the protein target. III. COLONY HYBRIDIZATION BACTERIAL DEFENSE AND GENETIC ENGENEERING • Use of segments of single-stranded nucleic acids whose identity is already known (labeled probes) in detecting specific gene • • • • A bacterium uses a restriction enzyme to defend against bacterial viruses called bacteriophages, or phages. When a phage infects a bacterium, it inserts its DNA into the bacterial cell so that it might be replicated. The restriction enzyme prevents replication of the phage DNA by cutting it into many pieces. Restriction enzymes were named for their ability to restrict, or limit, the number of strains of bacteriophage that can infect a bacterium.