27Recombinant DNA Technology

27Recombinant DNA Technology - Recombinant DNA Technology...

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Unformatted text preview: Recombinant DNA Technology Introduction Recombinant DNA technology involves the use of in vitro molecular techniques to isolate and manipulate DNA fragments. Recombinant DNA molecules are DNA fragments that are covalently linked in the lab. This technology has enabled researchers to study the relationship between genes and phenotype, and understand gene structure and function. Applications of these technologies include gene therapy and the production of transgenic plants. Gene Cloning General information Molecular biologists frequently focus on the structure and function of proteins, or the genes that encode them. Gene cloning involves the isolating and making of many copies of a gene. Table 18.1 summarizes some of the uses of gene cloning. Cloning experiments may involve two kinds of DNA molecules: chromosomal DNA and vector DNA. going to replicate t he dna we're interested in as well as the bacterial dna A vector is a small piece of DNA into which a gene of interest is introduced. The vector is then placed within a living cell, where it replicates and produces multiple identical copies of the inserted gene (Table 18.2). The vector acts as the carrier of the gene to be studied. The cell that holds the vector is called the host cell. Chromosomal DNA is usually used as the source of the gene of interest. Vectors are usually derived from plasmids. the vector could be a v irus or more often a plasmid Plasmids are small circular pieces of DNA that are found in bacterial and some eukaryotic cells. Plasmids that contain genes that confer resistance to toxic compounds and antibiotics are called R factors. 1 bacterial artifi cial c hromosome yeast artifi cial chromosome Plasmids contain origins of replication that are recognized by the host cell. Many plasmids contain selectable markers, which enable the cell containing the plasmid to grow in the presence of an antibiotic or other toxic substance. A viral vector is a virus that contains a chromosomal gene. Other vectors, such as cosmids, BACs, and YACs, are described later in the course. Enzymes are used to cut DNA into pieces and join the pieces together. occur naturally, mainly in bacteria and they are a defense mechanism Enzymes called restriction endonucleases, or restriction enzymes, are used to cut DNA. Restriction enzymes recognize base sequences and then cleave the DNA at two defined locations (Figure 18.1). They are found in many bacterial species, where they protect the organism from the invasion of foreign DNA, particularly bacteriophages. AN-NA nucleotide sequence 5'->3' is the same in the two Examples different strands reads the same in both directions of some restriction enzymes, their sources, and recognition sequences are provided in Table 18.3. Restriction enzymes recognize palindromic sequences. that they each palindrome is cut bySome restriction enzymes produce fragments with “sticky ends,” which means a DIFFERENT restriction will hydrogen bond with each other due to their complementary sequences. enzyme make an asymmetric cut, t he ends are c omplementary to one another This allows for temporary (although unstable) interactions between DNA fragments. Permanent connections must be established by a DNA ligase. Gene cloning involves the insertion of DNA fragments into vectors, which are then propagated within host cells. The general process of gene cloning is presented in Figure 18.2. want a plasmid with ONE restriction site. want to open the plasmid, put DNA in there and close it up Chromosomal DNA is isolated and cut with a restriction enzyme. The plasmid vector is cut with the same restriction enzyme to generate sticky ends. The chromosomal and plasmid DNA are incubated together. DNA ligase is added to covalently link the chromosomal and plasmid DNA. It is called a recircularized vector if it only contains plasmid DNA. It is called a hybrid vector if it contains both plasmid and chromosomal DNA. 2 going to take cells that don't have any plamsmids in them because we want them to t ake up the one we have plasmid has a gene for resistance to ampicilin bacterial cell we're using is s ensitive to ampicillin, but plamsmid is resistant if we put bacterial cell that didnt take up plasmid and put on ampicillin, it dies if it took up the plasmid -- > plasmid has ampR resistant gene; anything that grows t ook up plasmid is it the non recombinant or t he one that took up the gene? Lac Z present ->B galac is present -> it will cleave X-gal, and you get blue color...for non-recombinant lac Z not present ->B galac not present, it will not cleave X-gal... no blue color... for plasmid + chromosome (recombinant) The DNA is mixed with living bacterial cells that have been treated with agents that make them permeable to DNA. These cells, called competent cells, can take up DNA from the extracellular medium. This is called transformation. Note: With a viral vector it is called transfection. Selectable markers allow researchers to eliminate bacterial cells carrying no vector at all, and to distinguish between bacterial cells carrying recircularized vectors and those carrying hybrid vectors. The bacterial cells used in the experiment are sensitive to ampicillin. But, the plasmid is carrying a gene for resistance to ampicillin (ampR). The plasmid also has a lacZ gene, which encodes !-galactocidase. The restriction site is within the lacZ gene. After the mixing process, the cells are plated on media containing ampicillin, IPTG (an inducer of the lacZ gene), and X-Gal. Cells that did not take up plasmids do not survive because they are still sensitive to ampicillin. Cells that did take up plasmids survive because the plasmid contains the ampR gene. X-Gal is a colorless compound that, when cleaved by !-galactocidase, becomes a blue dye. If a bacterial cell contains a recircularized plasmid (i.e., one without any chromosomal DNA), the lacZ gene will still be functional and will produce !galactocidase when induced by IPTG. A colony derived from such a cell will be blue on the culture plate. If a bacterial cell contains a hybrid plasmid (i.e., one with chromosomal DNA inserted into it), the lacZ gene will be disrupted and !-galactocidase will not be produced. A colony derived from such a cell will be clear on the culture plate. Host cells carrying hybrid vectors may then be isolated. These will need to be screened to determine which carries the gene of interest. Cohen, Chang, Boyer, and Helling if it grows on culture plate, t hat means it has plasmid, it has ampicillin resistant geene if blue, it has plasmid that is t he recircularized vector you s tarted with if colony is clear, it is a hybrid vector that is in the the first gene cloning experiment, c ell, has split lac Z Experiment 18A. In R gene, no inserted a kanamycin gene into a plasmid vector. B galac, no cleaving of Xgal, no blue color Several important discoveries led to the discovery of gene cloning. Khorana (1970) discovered that DNA ligase could covalently link DNA fragments. 3 plasmids dont always have resistance genes, we can engineer resistance genes into plasmids Mertz, Davis, and Sgaramella (1972) discovered that some restriction enzymes (e.g., EcoR1) produced sticky-end DNA. Cohen, Chang, Boyer, and Helling realized it might be possible to create recombinant DNA molecules by digesting DNA with EcoR1, allowing the fragments to hydrogen bond with each other, and then covalently linking the fragments together with DNA ligase. For Cohen, Chang, Boyer, and Helling to successfully clone a gene, they needed to identify a vector that could independently replicate itself once inside a host cell; they chose a plasmid vector. In their collection of plasmids, they had one small plasmid (pSC101), which had a single EcoR1 site and carried a gene for tetracycline resistance (tetR). They reasoned that they should be able to open up the plasmid with EcoR1 and insert another piece of DNA into this site. As a source of the gene to be inserted, they obtained a second plasmid (pSC102), which carried a gene for resistance to the antibiotic kanamycin (kanR). This plasmid had three EcoR1 sites, so cutting with the restriction enzyme would yield three fragments, one of which would, hopefully, carry an intact kanR gene. hypothesis A piece of DNA carrying a gene can be inserted into a plasmid vector using recombinant DNA techniques. If this recombinant plasmid is introduced into a bacterial host cell, it will be replicated and transmitted to daughter cells, producing many copies of the recombinant plasmid. small plasmids are good becuase we need to get them into a living bacterial cell, has t o get across cell wall and c ell membrane goign to cut up 102 and try to get a piece of 102 into 101. and because it's only a part of 102, we should be able to t ell them apart The Testing the hypothesis (Figure 18.3) Starting materials: Three different strains of E. coli: one strain that carried no plasmid, one that carried pSC101, and one that carried pSC102. three strains: one has plasmid 101, one has plasmid 102, one carrying no plasmid Grow the bacterial cells containing the plasmids. Break open the cells. Isolate each type of plasmid DNA by density gradient centrifugation. isolating the plasmids now have purifi ed plasmids Digest the plamid DNAs with EcoR1. Mix the two samples together. place into diff. tubes and add EcoR1 in 101 it just opens up the rings for 102 it makes 3 fragments f rom each plasmid 1/3 are going to have kan gene Add DNA ligase to covalently link DNA pieces. 4 possibilities: 101 just closed up 101 with 102 that does have k an or 101 with 102 that does NOT have kan Grow an E. coli strain that does not carry a plasmid and treat the cells with CaCl2 to make them permeable to DNA. Add the ligated DNA samples (see the step two steps above) to the bacterial cells. Most cells will not take up any plasmid, but occasionally a cell will take up one plasmid. Plate the cells on growth media containing both tetracycline and kanamycin and grow overnight to allow growth of visible bacterial colonies. Pick four colonies and grow in liquid culture containing radiolabeled deoxyribonucleotides. The plasmid in these colonies is designated pSC105. To isolate the radiolabeled plasmid DNA, break open the cells and subject the DNA to cesium chloride density gradient centrifugation. Collect fractions and count the amount of radioactivity in each fraction. As a control, subject pSC101 and pSC102 to the same procedures. Digest the plasmid DNAs with EcoR1, and subject them to gel electrophoresis. data and interpreting the data. In the control experiment, the pSC101 and pSC102 plasmids were mixed together and then subjected to cesium chloride density gradient centrifugation. This yielded two peaks. The pSC102 plasmid is larger than pSC101 and sediments at a density of 39.5 S, where as pSC101 sediments at 27 S. The plasmids isolated from a bacterial colony that was resistant to both tetracycline and kanamycin, pSC105, had an intermediate density of 32 S, indicating that this bacterial colony contained a recombinant plasmid, not a mixture of pSC101 and pSC102 plasmids. This was confirmed with gel electrophoresis. When digested with EcoR1, pSC101 yielded a single band and pSC102 yielded three bands. A mixture of pSC101 and pSC102 digested with EcoR1 yielded four bands. When pSC105 was digested with EcoR1, it yielded two bands, one corresponding to pSC101 and the other corresponding to the pSC102 fragment carrying the kanR gene. This experiment showed it is possible to create recombinant DNA molecules and to propagate them in bacterial cells. put on plates with antibiotics f or both tetra and kan only cells that will grow will be the s o when we analyze it we c an see things 102 is larger than 101, so it has a larger density 105 has an intermediate density and it's just one peak tells us: cells that grew The on t he culture containing kan and tetra didn't just take up t wo different plasmids (the t wo original plasmids)... if it did take up two separate plasmids, it would have two peaks... instead we only see one peak... it is a recombinant plasmid that has piece of 102 that had k an gene 105 contained the 101 and t he 102 fragment that had t he kan gene (in lane 4) 5 bacterial cell we put recombinant into, each c ell that replicated after it is a clone eukaryotic because ofDNA ca poly A tail at 3' end so we can make primer (piece of DNA) that will pair with it make polyT and it will bind to polyA c omplementry DNA doesn't have any introns in it can be made from mRNA via reverse transcriptase. The enzyme reverse transcriptase can use RNA as a template to create a complementary strand of DNA. This enzyme is present in retroviruses. The DNA made in this manner is called complementary DNA, or cDNA (Figure 18.4). cDNA lacks introns. This makes it simpler to insert cDNA into vectors for cloning. Restriction mapping is used to locate the restriction sites within a vector. if all you're doing is c utting open a circle in t he same place, that means there is only going to be one site Restriction enzyme sites on a piece of DNA can be mapped by cutting the DNA with different combinations of restriction enzymes and visualizing the cut DNA by gel electrophoresis. map. copies of DNA. ECOr1 cuts in one Figure 18.5 outlines the procedure to create a restriction place, BAM H1 cuts in one place = two bands Polymerase chain reaction (PCR) can be used to make many far apart on gel means c lose together closer you get to eachother on the gel, means farther apart Another mechanism of copying DNA, without cloning, is the use of the polymerase chain reaction (PCR – Figure 18.6). Template DNA contains the section of DNA to be copied. Primers define the region of the DNA (gene) to be copied. dNTPs provide materials for the synthesis of DNA. A crucial component of the procedure is the use of a thermostable DNA polymerase, called Taq polymerase, which was isolated from the bacterium Thermus aquaticus. The process involves denaturation of the DNA, annealing of the primers to the DNA, and synthesis of a new DNA strand. The procedure is conducted in a thermocycler, which provides precise timing and temperature controls. PCR can also be used to detect and quantify the amount of specific RNA in a living cell. This is called reverse-transcriptase PCR, or RT-PCR. 6 Detection of Genes, Gene Products, and Protein-DNA Interactions General information G prokarotyic organisms don't ene detection refers to methods that distinguish one particular gene among a mixture of have an introns, so if we want thousands of other genes. t o put a human mRNA into a prokaryotic cell, we must use This can also involve the detection of RNA from a particular gene. an mRNA that has been processed, so we must used library is constructed and then screened by colony hybridization to identify a cloned A DNA a c DNA take mRNA that has been processed for a gene use cDNA that is c omplemetnary put into plasmid put that into bacterial cell gene. A collection of DNA hybrid vectors is called a DNA library. If the starting material was chromosomal DNA, it is called a genomic library. If the starting material was cDNA, it is a cDNA library. cDNA libraries are useful if a researcher wants to express the encoded protein of interest in a cell that would not splice out introns correctly. The construction of a DNA library is outlined in Figure 18.7. A DNA probe may be used to identify a colony that contains the gene of interest. This process is called colony hybridization (Figure 18.8). put in a radioactive probe that is complementary to what we are looking for take membrane and put onto x ray fi lm, radioactivity will expose what we are looking f or The probe, a single-stranded DNA, may already be available from a cloned gene, or a similar gene may be used from another species. Amino acid sequence analysis of the protein produced by the gene may also be used to generate probes. The purified protein can be used to generate antibodies, which can be labeled and used as probes. Southern blotting is used to detect DNA sequences. Southern blotting, invented by Edwin Southern (1975), can be used to detect the presence of a specific gene in a mixture of chromosomal DNA fragments. It can also be used to: Determine the copy number of the gene within the genome. Detect small deletions in a gene. Identify gene families. Identify homologous genes among different species (called a zoo blot). 7 The experimental procedure for Southern blotting is shown in Figure 18.9. Prior to Southern blotting the gene of interest (or a fragment of it) is cloned. The cloned DNA is then labeled (e.g., radiolabeled) in vitro, and the labeled DNA is used as a probe to detect the presence of the gene or a homologous gene within a mixture of many DNA fragments. The chromosomal DNA is digested with a restriction enzyme. The chromosomal pieces are run on a gel that separates them according to size. The bands within the gel are transferred to a nylon membrane in a step known as blotting (Figure 18.9b,c). The radioactive probe DNA is used to detect the presence of the complementary pieces of DNA on the filter. The experimental procedure can be varied in temperature and ionic strength. Temperature variations determine at what level of similarity the probe will bind with the DNA. At high temperature, the DNA must be almost identical. This is called high stringency. At low temperature, the probe does not need to be an exact complement. This is called low stringency. Low stringency may be used to detect gene families. t ells you if gene is t urned off or on, if mRNA is found then it is turned ON. if there is less (band is higher up and not as thick) t hen it means it is processed differently and not as much RNA Northern blotting is used to detect RNA. Northern (or reverse-Southern) blotting is used to detect a specific RNA among a mixture of many RNA molecules. It is used to study the transcription of genes at the molecular level. It is useful to determine if a gene is active in a particular cell type, or at a specific stage of development. The experimental system is similar to Southern blotting. The probe is usually a fragment of DNA from the cloned gene. An example of the results are shown in Figure 18.10. Western blotting is used to detect protein. Western blotting determines if a specific protein is being made in a certain cell type, or at a particular stage of development. 8 probe is an antibody The process is similar to a Southern blot. The proteins must be treated with SDS (sodium dodecyl sulfate) to denature the protein and coat them with negative charges. t hings that are lighter move farther, heavier move less distance The proteins are run out on a SDS-PAGE gel. PAGE stands for polyacrylamide gel electrophoresis. Western blots use antibodies as probes. Antibodies bind to sites on molecules called epitopes (antigens). An antigenic site on a protein is a short sequence of amino acids. The primary antibody binds to the epitope, and then a second labeled antibody binds to the first. The secondary antibody allows the researcher to detect the protein of interest on the gel (Figure 18.11). Techniques can be used to detect the binding of proteins to DNA sequences Researchers may also want to study the binding of proteins to specific sites on the DNA. Gel retardation assays and DNA footprinting procedures make this possible. Gel retardation assays, also called band shift assays, are shown in Figure 18.12. The binding of a protein to the DNA slows its migration through the gel. DNAse I footprinting (Figure 18.13) attempts to identify the regions of the DNA that interact with a DNA-binding protein. Involves the molecular interactions of the DNA, the DNA-binding protein, and agents that alter the DNA structure. Bound proteins protect DNA from DNAse I degradation. Analysis and Alteration of DNA Sequences General information DNA sequencing enables researchers to determine the base sequence of DNA. Site-directed mutagenesis allows researchers to change the sequence of DNA in cloned sequences. DNAse I chops up DNA what we're going to see on the gel is the ones with t he radioactive piece on t he end of it so radioactive pieces will t ravel farther, others will be up at the top 9 c omplementary s equence, band by band, you have the s equence...called aThe s equence ladder dideoxy method of DNA sequencing is based on our knowledge of DNA replication. The process uses dideoxyribonucleotides that lack the 3’–OH group (Figure 18.14). This prevents the synthesis of the DNA strand during a PCR reaction. This is called chain termination. The process of dideoxy sequencing is illustrated in Figure 18.15. The output of the method is either a sequence ladder (Figure 18.15) or a computerized readout from automated sequencing (Figure 18.16). Site-directed mutagenesis is a technique to alter DNA sequences. The analysis of mutations can provide useful information about genetic processes, gene expression, and phenotype. Site-directed mutagenesis allows a researcher to introduce mutations into cloned genes or other DNA segments. This process is shown in Figure 18.17. Please see the Conceptual Summary and Experimental Summary for Chapter 18 on pages 507 and 508. This lecture outline was prepared from Genetics: Analysis and Principles, by Brooker, 2009 (3rd edition). It contains phrases and entire sentences taken verbatim from that source, and is in no way meant to represent original work by Mark Bierner. 10 ...
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