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Bio.203-20.Biotech
Molecular Biology Ch. 20 - Biotechnology
| Question | Answer |
|---|---|
| Recombinant DNA | DNA molecules formed when segments of DNA from two different sources (e.g. species) are combined in vitro (in a test tube). |
| Biotechnology | The manipulation of organisms or their components to make useful products, e.g. selective breeding of farm animals, wine production, etc. |
| Genetic engineering | The direct manipulation of genes for practical purposes. |
| In eukaryotic genomes, genes occupy what proportion of the chromosomal DNA? | Only a small portion; the rest being noncoding nucleotide sequences. |
| Plasmids | E. coli and many other bacteria have plasmids: small circular DNA molecules that replicate separately from the bacterial chromosome. |
| The significance and importance of plasmids in bacteria? | A plasmid only has a small number of genes; these genes may be useful when the bacterium is in a particular environment but may not be required for survival or reproduction under most conditions. |
| Process of DNA cloning in bacteria overview, step: (1) | Isolate plasmid from a bacterium and genetically engineer it for efficient cloning. Then insert DNA from another source into the plasmid. The result: recombinant DNA molecule. |
| Process of DNA cloning in bacteria overview, step: (2) | Insert the plasmid (now a recombinant DNA molecule) into a bacterium. The result: a recombinant bacterium is produced. |
| Process of DNA cloning in bacteria overview, step: (3) | The host cell is grown in culture to form a clone of cells containing the cloned genes of interest. The cloned genes are being expressed, producing proteins of interest. |
| Process of DNA cloning in bacteria overview, step: (4) | The copied genes are extracted for research/application and the copied proteins are harvested for research/application. |
| Examples of applications for copied genes | (1) Gene for pest resistance inserted into plants; (2) Gene used to alter bacteria for cleaning up toxic waste. |
| Examples of applications for harvested proteins from cloned genes | (1) Protein dissolves blood clots in heart attack therapy; (2) Human growth hormone treats stunted growth. |
| Definition of gene cloning | The production of multiple copies of a single gene. |
| Gene cloning is useful for two basic purposes: | (1) to make many copies of, or *amplify*, a particular gene and (2) produce a protein product. |
| Restriction enzyme | AKA Restriction endonuclease. It is a type of enzyme that recognizes and cuts DNA molecules foreign to a bacterium (e.g. phage genomes), thus protecting the cell (restricting viral proliferation). The enzyme cuts at specific nucleotide sequences. |
| Endonucleases | Enzymes that cut DNA molecules at a limited number of specific locations. |
| Are restriction enzymes found in eukaryotes? | Eukaryotes don't have restriction enzymes, although they do have other types of endonucleases. E.g. topoisomerase has endonuclease activity. |
| Restriction site | Each restriction enzyme is very specific, recognizing a particular short DNA sequence called the restriction site. The enzyme cuts both DNA strands at precise points within the restriction site. |
| How is the DNA of a bacterial cell protected from the cell's own restriction enzymes? | By the addition of methyl groups (—CH3) to adenines or cytosines within the sequences recognized by the enzymes. |
| Overview of using a restriction enzyme to make recombinant DNA, Step: (1) | A restriction enzyme cuts the sugar-phosphate backbones of DNA at specific regions, breaking apart the DNA and creating one fragment. The ends of the fragment appear staggered (the antiparallel strands extend farther than their complementary strands). |
| Overview of using a restriction enzyme to make recombinant DNA, Step: (2) | DNA fragments from other sources will join with the fragment just created by base pairing at their sticky ends. The sticky ends are the staggered ends of the fragments created when the restriction enzyme cut the DNA. |
| Overview of using a restriction enzyme to make recombinant DNA, Step: (3) | DNA ligase will seal up the connected strands. |
| Restriction fragments | The name given to the DNA segments that result from the cutting of DNA by a restriction enzymes. In circular DNA the number of resultant fragments equals the number of restriction sites. In linear DNA that is not the case. |
| Cloning vector | The original plasmid is called a cloning vector. Defined as a DNA molecule that can carry foreign DNA into a host cell and replicate there. |
| Why are bacterial plasmids so widely used as cloning vectors? | They can be readily obtained from commercial suppliers, manipulated to form recombinant plasmids by insertion of foreign DNA in vitro, and then introduced into bacterial cells. Bacterial cells also multiply rapidly which increases efficiency. |
| Steps in cloning a hummingbird's genomic DNA step: (1) | Isolate the bird's genomic DNA and obtain our chosen cloning vector, a genetically engineered plasmid. The plasmid has been engineered to carry two genes: AmpR (ampicillin resistance gene) and lacZ (which encodes the enzyme β-galactosidase). |
| Steps in cloning a hummingbird's genomic DNA step: (2) | Cut both DNA samples with the same restriction enzyme. The plasmid was engineered to only have one restriction site so it will only be cut once (note, the lacZ gene is contained within the plasmid's restriction site). The bird DNA will be cut many times. |
| Steps in cloning a hummingbird's genomic DNA step: (3) | Mix the cut plasmids and bird DNA fragments. Some join (at the sticky ends) by base pairing; add DNA ligase to seal them together. The result: many recombinant plasmids and many nonrecombinant plasmids. |
| Steps in cloning a hummingbird's genomic DNA step: (4) | The recombinant plasmids are then added to a solution of bacteria that have mutations in their own DNA in the lacZ gene making them unable to hydrolyze lactose. The bacteria take up the foreign DNA by transformation. |
| Steps in cloning a hummingbird's genomic DNA step: (5) | Plate the recombinant bacteria on nutrient-containing agar medium supplemented with ampicillin and X-gal (a synthetic molecule hydrolyzed by β-galactosidase; turns blue when hydrolyzed). Incubate until colonies grow. |
| Steps in cloning a hummingbird's genomic DNA step: (6) | Only a cell that took up a plasmid, which has the AmpR gene, will reproduce and form a colony. Colonies with nonrecombinant plasmids will be blue because they can hydrolyze X-gal (i.e. the plasmids that didn't get cut by the restriction enzyme are blue). |
| Steps in cloning a hummingbird's genomic DNA: RESULTS | We have produced a culture of plasmids that (if they're not blue) have each taken up a fragment of the bird's DNA. Taken together, the non-blue colonies should represent all the DNA sequences from the hummingbird genome. |
| What is the cloning procedure outlined in the hummingbird example known as? | The "shotgun" approach because no single gene is targeted for cloning. |
| Genomic library | The complete set of plasmid-containing cell clones, each carrying copies of a particular segment from the initial genome. Each "plasmid clone" in the library is like a book containing specific information. |
| Are any other entities (other than bacteria) used as cloning vectors? | Bacteriophages have been used, but not very commonly. Fragments are inserted into the viral DNA, then the virus infects a colony and expresses the DNA. Another type of vector widely used is a bacterial artificial chromosome (BAC). |
| Bacterial artificial chromosome (BAC) | A large plasmid that acts as a bacterial chromosome and can carry inserts of 100,000 to 300,000 base pairs (100 - 300 kb). |
| The pros and cons of using BACs | The very large insert size minimizes the number of clones needed to make up the genomic library, but it also makes them more challenging to work with in the lab. The insert may be later cut up into smaller pieces that are "subcloned" into plasmid vectors. |
| Review: Each "book" in a genomic library is... | ... a clone of a bacterial cell containing a copy of a particular foreign genome fragment in its recombinant plasmid. |
| How are clones stored? | They're stored in "multiwell" plastic plates, with each clone occupying one well. The library of an entire genome requires many such plates. |
| cDNA library | A gene library containing clones that carry complementary DNA (cDNA) inserts. The library includes only the genes that were transcribed in the cells whose mRNA was isolated to make the DNA. |
| cDNA | A double-stranded DNA molecule made in vitro using mRNA as a template and the enzymes reverse transcriptase and DNA polymerase. A cDNA molecule corresponds to the exons of a gene. (Introns are not included). |
| Creating a cDNA molecule, step: (1) | Reverse transcriptase is added to a test tube containing mRNA from the certain type of cell. |
| Creating a cDNA molecule, step: (2) | Reverse transcriptase makes the first DNA strand using the mRNA as a template and a stretch of thymine deoxyribonucleotides (dT's) as a DNA primer. |
| Creating a cDNA molecule, step: (3) | mRNA is degraded by another enzyme. |
| Creating a cDNA molecule, step: (4) | DNA polymerase synthesizes the second strand, using a primer in the reaction mixture. (Several options exist for primers.) |
| Creating a cDNA molecule, step: (5) | The result is cDNA, which carries the complete coding sequence of the gene, but no introns. |
| Where does the thymine deoxyribonucleotide primer attach to the mRNA to be reverse transcribed? | To the poly-A tail at the end of the mRNA which was added during RNA processing. |
| How to make a cDNA library | Modify the cDNA molecules by adding restriction enzyme recognition sequences at the ends. Then the cDNAs will be inserted into vector DNA molecules. The cDNAs that are cloned will make up the cDNA library. |
| When would you prefer a genomic library to a cDNA library? | If you want to clone a gene but you don't know what cell type expresses it. Also, if you are interested in the regulatory sequences or introns associated with a gene (as the cDNA molecules are composed only of exons). |
| When would you prefer a cDNA library to a genomic library? | If you want to study a specific protein a cDNA library made from cells expressing the genes is ideal. Or if you want to study sets of genes in particular cells types (liver cells). Or if you want to trace the same cell type over an organism's life cycle. |
| Nucleic acid hybridization | The process of base pairing between a gene and a complementary sequence on another nucleic acid molecule. The complementary molecule, the nucleic acid probe, can be DNA or RNA. |
| Nucleic acid probe | A labeled single-stranded nucleic acid used to locate a specific nucleotide sequence in a nucleic acid sample. The probe bonds to the complementary sequence wherever it occurs. Rad. isotope, fluorescent, etc labels on the probe can be used to track it. |
| Example of how to use a nucleic acid probe to detect a sequence of DNA | You have created a genomic library but you don't know exactly what the sequence of the gene of interest is; you only know a portion of it. Solution: Create nucleic acid probe to bond with the portion you do know, track it to locate the entire gene. |
| Steps to detecting a specific DNA sequence by hybridization with a nucleic acid probe, step: (1) | Transfer cells from each plate/well from your genomic library into a defined spot on a special nylon membrane. The membrane is treated to break open the cells and denature their DNA. The resulting single stranded DNA molecules stick to the membrane. |
| Steps to detecting a specific DNA sequence by hybridization with a nucleic acid probe, step: (2) | The membrane is then incubated in a solution of radioactive probe molecules complementary to the gene of interest. The probes now base pair with DNA molecules. |
| Steps to detecting a specific DNA sequence by hybridization with a nucleic acid probe, step: (3) | The membrane is laid under photographic film; radioactive areas expose the film. Black spots correspond to the locations of the DNA that has hybridized to the probe. Each spot is traced to the original well containing the bacterial clone with the gene. |
| One way nucleic acid probes are created | By taking portions of an identified gene, then using it as a probe to identify similar genes. |
| Expression vector | A cloning vector that contains a highly active bacterial promoter just upstream of a restriction site where a eukaryotic gene can be inserted, allowing the gene to be expressed in a bacterial cell. |
| Problem: bacterial cells can't understand eukaryotic sequences that contain long strips of introns because they don't have RNA-splicing machinery. Solution? | Use a cDNA form of the gene (includes only exons). |
| Molecular biologists can avoid eukaryotic-bacterial incompatibility by... | ... using eukaryotic cells such as yeasts, rather than bacteria, as hosts for cloning and/or expressing eukaryotic genes of interest. |
| Two advantages of using yeasts (single-celled fungi) | (1) They are as easy to grow as bacteria, and (2) they have plasmids, a rarity among eukaryotes. Note: scientists have even created recombinant plasmids that combine yeast/bacterial DNA and can replicate in either type of cell. |
| Another reason to use eukaryotic cells (relating to RNA processing) | Many eukaryotic proteins will not function unless they are modified after translation, for example, by glycosylation or the addition of lipid groups. Bacterial cells cannot carry out RNA processing. |
| Electroporation | A method used for introducing recombinant DNA into eukaryotic cells. A brief electrical pulse applied to a solution containing cells created temporary holes in their plasma membranes, through which DNA can enter. |
| how to manually introduce recombinant DNA into eukaryotic cells | By using microscopically thin needles to inject DNA directly into single eukaryotic cells. |
| To get DNA into plant cells... | ... The soil bacterium Agrobacterium can be used. |
| Polymerase chain reaction (PCR) | A technique for amplifying DNA in vitro by incubating it with specific primer, a heat-resistant DNA polymerase, and nucleotides. |
| Benefits of using PCR? | It's quicker and more selective than DNA cloning. A specific target segment is quickly amplified. |
| Summary of the PCR process | During each cycle the reaction mixture is heated to denature (separate) the DNA strands, then cooled to allow annealing (hydrogen bonding) of primers to the strands. A special heat-resistant DNA polymerase extends the primers in 5' to 3' direction. |
| In PCR what is the DNA polymerase that is heat-resistant enough to be used? | Taq polymerase, isolated from the bacterial species Thermus aquaticus, a species of bacteria native to hot springs. I.e. natural selection resulted in a heat-stable polymerase. |
| Polymerase Chain Reaction, step: (1) | Denaturation: heat briefly separate DNA strands |
| Polymerase Chain Reaction, step: (2) | Annealing: Cool to allow primers to form hydrogen bonds with ends of the target sequence. |
| Polymerase Chain Reaction, step: (3) | Extension: DNA polymerase adds nucleotides to the 3' end of each primer. |
| Polymerase Chain Reaction, step: (4) | In cycle 3, 8 molecules are yielded, 2 of which match the target sequence (same sequence AND appropriate length). |
| After the third cycle of PCR (which yielded 2 target molecules) how can you calculate the output of the successive cycles? | The number of target molecules equals 2^n where n = the number of cycles beginning at the third cycle. e.g. the fifth PCR cycle will yield 2^3 = 8. The 32nd cycle will yield 2^30 = 10^9 (over 1bil) target molecules. |
| Why can't PCR amplification substitute for gene cloning altogether? | Occasional errors during PCR replication impose limits on the number of good copies that can be made. When dealing with large amounts of genes PCR should not be used. |
| When PCR is used to provide a specific DNA fragment for cloning, how are the errors dealt with? | The resulting clones are sequenced to select clones with error-free inserts. Note: PCR errors also impose limits on the length of DNA fragments that can be copied. |
| Gel electrophoresis | A technique for separating nucleic acids or proteins on the basis of their size and electric charge, both of which affect their rate of movement through an electric field in a gel made of agarose or another polymer |
| On a molecular level, how does gel electrophoresis work? | Nucleic acids carry negative charge on their phosphate groups so they all travel toward the positive pole in an electric field. As they move, the thicket of agarose fibers impedes longer molecules more than it does shorter ones, separating them by length. |
| After the molecules are separated, what is observed? | The DNA molecules are separated into bands in the gel, each band consisting of many thousands of DNA molecules of the same length. |
| Overview of restriction fragment analysis using gel electrophoresis | DNA fragments produced by restriction enzyme digestion are separated by gel electrophoresis. The resultant band-pattern gives you information about the number and sizes of fragments. |
| Polymorphisms | Variations in DNA sequence among a different DNA molecules, e.g. between alleles of a particular gene. |
| Restriction fragment length polymorphism (RFLP, pronounced "rif-lip") | A single nucleotide polymorphism (SNP) that exists in the restriction site for a particular enzyme, thus making the site unrecognizable by that enzyme and changing the lengths of the restriction fragments formed by digestion with that enzyme. |
| Example of a restriction fragment length polymorphism | Sickle-cell disease is caused by a mutation of a single nucleotide located within an RFLP in the human β-globin gene. For many years restriction fragment analysis was used to distinguish normal and sickle-cell alleles. |
| How do RFLPs allow gel electrophoresis to distinguish normal and sickle-cell alleles? | Normal cells have two restriction sites within the β-globin gene recognized by the DdeI restriction enzyme. The sickle-cell allele only has one restriction site. Therefore the normal allele produces three bands while the sickle-cell allele produces two. |
| If you wanted to identify whether someone is heterozygous for the sickle-cell allele, how can you do that? | If you tried gel electrophoresis you would produce very many bands and would not be able to distinguish which bands contain the allele and which ones do not. Southern blotting would be necessary. |
| Southern blotting | A technique that enables specific nucleotide sequences to be detected in samples of DNA. It involves gel electrophoresis of DNA molecules and their transfer to a membrane (blotting), followed by nucleic acid hybridization with a labeled probe. |
| Southern blotting example: screening for sickle-cell anemia, step (1) | DNA samples from patients are mixed into separate vials, each containing the same restriction enzyme: DdeI. Digestion of each sample yields a mixture of thousands of restriction fragments. |
| Southern blotting example: screening for sickle-cell anemia, step (2) | The restriction fragments in each sample are separated by gel electrophoresis, forming a characteristic pattern of bands. There are thousands of bands so nothing can be discerned with regard to the sickle-cell gene. |
| Southern blotting example: screening for sickle-cell anemia, step (3) | Blotting: Capillary action pulls the alkaline solution upward through the gel, denaturing and transferring the DNA to a nitrocellulose membrane. This produces a blot with a pattern of DNA bands exactly like that of the gel. |
| Southern blotting example: screening for sickle-cell anemia, step (4) | The nitrocellulose blot is placed into a plastic bag containing a solution full of radioactively labeled nucleic acid probes for the β-globin gene. The probes base pair with any restriction fragments containing portions of the β-globin gene. |
| Southern blotting example: screening for sickle-cell anemia, step (5) | A sheet of photographic film is laid over the blot. The radioactivity in the bound probe exposes the film to form an image corresponding to those bands containing DNA that base-paired with the probe. |
| Southern blotting example: screening for sickle-cell anemia, RESULTS | Examining the film you can deduce the no. of restriction sites each patient's genes contain. If a patient's alleles both contain one site: they are homozygous (diseased); if one allele has two while the other has one: heterozygous; if both = two: normal |
| DNA Sequencing | Determining the order of a molecule of DNA's nucleotide bases. |
| The first automated procedure for DNA sequencing | Dideoxyribonucleotide (or dideoxy) chain termination method. |
| Dideoxyribonucleotide (ddNTP) | A modified nucleotide which lacks a 3' -OH group, the site for attachment of the next nucleotide. Thus incorporation of a ddNTP terminates a growing strand of DNA. |
| Dideoxy chain termination method for sequencing DNA, step (1) | The DNA fragment to be sequenced is denatured into single strands and incubated in a test tube with the ingredients for DNA synthesis: a primer, DNA polymerase, the four deoxyribonucleotides, and the four dideoxyribonucleotides, each tagged a diff. color |
| Dideoxy chain termination method for sequencing DNA, step (2) | Synthesis of each new strand starts at the 3' end of the primer and continues until a dideoxyribonucleotide is inserted, at random, instead of a deoxyribonucleotide. This prevents further elongation of the strand. |
| Dideoxy chain termination method for sequencing DNA, step (3) | After all strands are generated, each of random length (because the colored dideoxyribonucleotides were inserted randomly), they're separated by passage through a polyacrylamide gel (shorter strands moving through quicker). Thus they're sorted by size. |
| Dideoxy chain termination method for sequencing DNA, step (4) | All of the copies of the original strand are now ordered by size. Because the dideoxyribonucleotides at the ends are colored, each color corresponding to their specific nucleotide, the sequence of the DNA can be determined. |
| Sequencing by synthesis | A chemical "trick" enables electronic monitors to identify which of the four nucleotides is added to a strand of DNA as it's synthesized, one nucleotide at a time. |
| Two methods of studying the expression of single genes | 1. Northern blotting, 2. Reverse transcriptase-polymerase chain reaction (TR-PCR) |
| Northern blotting | A technique that enables specific nucleotide sequences to be detected in samples of mRNA. It involves gel electrophoresis of RNA molecules and their transfer to a membrane (blotting), followed by nucleic acid hybridization with a labeled probe |
| How can northern blotting be used to study gene expression | Periodically carry out northern blotting on the mRNA molecules produced by an embryo at different stages of development. Examine the band-patters and identify whether a particular gene is being transcribed at different stages of development. |
| Reverse transcriptase-polymerase chain reaction (RT-PCR) | A technique for determining expression of a particular gene. It uses reverse transcriptase and DNA polymerase to synthesize cDNA from all the mRNA in a sample and then subjects the cDNA to PCR amplification using primers specific for the gene of interest |
| Describe in situ hybridization | A technique using nucleic acid hybridization with a labeled probe to detect the location of a specific mRNA in an intact organism |
| DNA microarray assays | A method to detect and measure the expression of thousands of genes at a time. Small amounts of a large number of single-stranded DNA fragments of different genes are fixed to a glass slide and tested for hybridization with samples of labeled cDNA |
| The microarray is also known as... | ... a "DNA chip" by analogy to a computer chip |
| Running a DNA Microarray assay, step: (1) | Isolate mRNA |
| Running a DNA Microarray assay, step: (2) | Make cDNA by reverse transcription, using fluorescent labeled nucleotides |
| Running a DNA Microarray assay, step: (3) | Apply the cDNA mixture to a microarray, a microscope slide which copies of single-stranded DNA fragments from the organism's genes are fixed, with a different gene in each spot. The cDNA hybridizes with complementary DNA on the microarray |
| Running a DNA Microarray assay, step: (4) | Rinse off excess DNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample. |
| Three methods of determining the function of genes | 1. In vitro mutagenesis 2. RNA interference (RNAi) 3. Genome-wide association studies |
| In vitro mutagenesis | A technique used to discover the function of a gene by cloning it, introducing specific changes into the cloned gene's sequence (i.e. knocking out the gene), reinserting the mutated gene into a cell, and studying the phenotype of the mutant |
| RNA interference (RNAi) as it is used to determine gene function | Uses the phenomenon of RNA interference (using synthetic RNA molecules matched to a particular gene sequence to trigger breakdown of the gene's mRNA or block its translation) selectively knock genes out and analyze their functions |
| Genome-wide association studies | A large-scale analysis of the genomes of many people having a certain phenotype or disease, with the aim of finding genetic markers that correlate with that phenotype or disease |
| Genetic markers | DNA sequences that vary in the population |
| Single-nucleotide polymorphism (SNP) | Among the most useful of genetic markers, an SNP is a single base-pair site in a genome where nucleotide variation is found in at least 1% of the population. |
| How are SNPs used as genetic markers? | In conducting a genome-wide association study, if two individuals, one affected by a particular disease and the other unaffected, are found to contain an SNP that is shared by one but not the other, researchers will focus on that region and sequence it |
| In the vast majority of cases, does the SNP turn out to have contributed to the disease? | No, in the majority of cases the SNPs are located in noncoding regions. |
| Organismal cloning | The production of one or more organisms genetically identical to the parent that donated the single cell. |
| Totipotent | Describing a cell that can give rise to all parts of the embryo and adult, as well as extraembryonic membranes in species that have them. I.e. mature cells can "dedifferentiate" and then give rise to all the specialized cell types of the organism |
| What kinds of organisms have totipotent cells? | Plant cells; plant cloning is now used extensively in agriculture. E.g. cloning orchids is the only commercially practical means of reproducing plants. |
| Can adult animal cells "dedifferentiate" like plant cells? | No, animal cells generally do not divide in culture, much less develop into the multiple cell types of a new organism. |
| Describe the experiment that tested the ability of animal DNA to develop in a less differentiated cell | J. Gurdon et al. transplanted frog nuclei from cells of different ages into embyros. Younger nuclear transplants were more likely to develop the embryo into adult organisms. Older nuclear transplants were not likely to do so. |
| Overview of the cloning of the sheep to produce Dolly | Donor cells are semistarved, arresting the cell cycle, causing dedifferentiation. The nucleus from the dedifferentiated cell is then transplanted into an enucleated egg cell. The early embryo was grown in culture, then implanted into the surrogate mother |
| Problems with animal cloning | Old transplanted DNA has problems: shorter telomeres; methylated DNA; etc. |
| Stem cell | A relatively unspecialized cell that can both reproduce itself indefinitely and, under appropriate conditions, differentiate into specialized cells of one or more types. |
| Stem cells can be isolated from early embryos at a stage (in humans) called the ____________ stage. | Blastocyst |
| Embryonic stem cells | Stem cells extracted from the embryos of organisms. |
| Blastocyst | A thin-walled hollow structure in early embryonic development that contains a cluster of cells called the inner cell mass from which the embryo arises. |
| Adult stem cells | They serve to replace nonreproducing specialized cells as needed. In contrast to ES cells, adult stem cells are unable to give rise to all cell types in an organism, though they can generate multiple types. |
| Examples of adult stem cells | Some adult bone marrow stem cells can generate all the different kinds of blood cells while others can differentiate into bone, cartilage, fat, muscle, etc. Some adult stem cells in the brain can produce certain kinds of nerve cells. |
| One application of our understanding of adult stem cells | Adult stem cells from bone marrow have long been used as a source of immune system cells in patients whose own immune systems are nonfunctional because of genetic disorders or radiation treatments for cancer. |
| Embryonic stem (ES) cells hold more promise than adult stem cells because ES cells are ______________ | Pluripotent |
| Pluripotent | Capable of differentiating into many different cell types |
| How are ES cells currently obtained for research? | They are currently obtained from embryos donated by patients undergoing infertility treatment or from long-term cell cultures originally established with cells isolated from donated embryos. |
| How might ES cell research be expedited in the future? | If it becomes possible to clone ES cells, obtaining large amounts of ES cells for research would become easier. |
| Therapeutic cloning | Cloning ES cells to treat diseases by producing cells to repair or replace damaged organs. ES cells aren't rejected by the host. |
| Induced pluripotent stem (iPS) cells | Researchers transformed fully differentiated skin cells into ES cells by using retroviruses (i.e. viral vectors) to introduce extra cloned copies of four "stem cell" master regulatory genes. |
| Are iPS cells exactly the same as ES cells extracted from the embryo? | Unfortunately no, research groups have uncovered differences between iPS and ES cells in gene expression and other cellular functions, such as cell division. |
| Two uses of iPS/ES cells | 1. Cells in diseased patients can be reprogrammed to become iPS cells which can then be studied in order to find potential treatments, 2. Regenerative medicine |
| Using DNA technology to diagnose HIV | Use RT-PCR to detect and amplify HIV RNA in blood or tissue samples |
| Using DNA technology to detect genetic disorders | Use PCR with primers that target genes with certain genetic disorders. The amplified DNA product is then sequenced to reveal the presence or absence of the disease-causing mutations |
| Why are SNPs harmful? | The presence of particular SNPs is correlated with an increased risk for Alzheimer's disease, heart disease, and some types of cancers. Genetic tests are available to detect SNPs. |
| Gene therapy | The introduction of genes into an afflicted individual for therapeutic purposes. |
| For gene therapy to be permanent... | ...the cells that receive the normal allele must be ones that multiply throughout the patient's life, such as bone marrow cells |
| Overview of gene therapy, step (1) | Patient presents with a disease, a severe combined immunodeficiency (SCID), meaning they can't produce a crucial enzyme due to a defective bone marrow cell gene. First step to curing them: clone a normal allele of the gene and insert it into a retrovirus |
| Overview of gene therapy, step (2) | Let the viral vector (retrovirus) infect bone marrow cells that have been removed from the patient and cultured. |
| Overview of gene therapy, step (3) | Viral DNA carrying the normal allele will insert into the chromosome |
| Overview of gene therapy, step (4) | The new cells, with the normal allele inserted into their DNA, will be injected into the patient who will now be able to produce the crucial enzyme |
| In the year 2000, gene therapy was used in a clinical trial to treat patients with SCID; what was the result? | While the patients initially responded favorably to the treatment, a large number of them got cancer soon after. This underlines the fact that gene therapy has not yet been perfected. |
| How has DNA technology helped the pharmaceutical industry? | Determining the sequence and structure of proteins has led to the identification of small molecules that combat certain cancers by blocking the function of these proteins. |
| Imatinib | A drug (trade name Gleevec) which is a small molecule that inhibits a specific receptor tyrosine kinase. The overexpression of this receptor, resulting from a chromosomal translocation, is instrumental in causing chronic myelogenous leukemia. |
| Tissue plasminogen activator (TPA) | Developed through genetic engineering methods, if it is administered shortly after a heart attack, TPA helps dissolve blood clots and reduces the risk of subsequent heart attacks. |
| How are human growth hormone and insulin made? | Genetically engineered cells both produce and secrete large amounts of the proteins. They are then used as replacement therapies for people lacking them, e.g. diabetes (insulin) and a form of dwarfism where the patient lacks HGH. |
| Protein production by "Pharm" animals | In some cases, instead of using cell systems to produce large quantities of protein products, pharmaceutical scientists can use whole animals. They can introduce a gene from an animal of one genotype into the genome of another, often another species. |
| Transgenic animals | Pertaining to an organism whose genome contains a gene introduced from another organism of the same or a different species. |
| How to create transgenic animals | 1. Remove eggs from recipient species, fertilize them in vitro, 2. Inject cloned donor DNA into the nuclei of the fertilized eggs, 3. The cells integrate the DNA "transgene", now expressing the genes, 4. Implant engineered embryo into surrogate mother |
| Example of the use of pharm animals | A transgene for a human blood protein such as antithrombin can be inserted into the genome of a goat in such a way that the transgene's product is secreted in the animal's milk. The protein is then purified from the milk. |
| Genetic profile | An individual’s unique set of genetic markers, detected most often today by PCR or, previously, by electrophoresis and nucleic acid probes. |
| The first use of DNA technology in criminal investigations | The FBI conducted RFLP analysis by Southern blotting to detect similarities and differences in DNA samples. This method required about 1,000 cells as evidence, obtained from the crime scene. This began in 1988. |
| Modern forensics | Today, forensic scientists use a more sensitive method that takes advantage of variations in length of genetic markets called short tandem repeats (STRs) |
| Short tandem repeats | DNA contains multiple tandem repeated units of two to five nucleotides. Variations in STRs act as genetic markers in STR analysis, used to prepare genetic profiles. PCR and electrophoresis is used to determine the number of repeats. |
| Why does the FBI prefer examining STRs rather than entire genetic profiles? | Southern blotting isn't required so it's quicker than RFLP analysis. Also the PCR step allows use of the method even when the DNA is in poor condition or available only in minute quantities. A tissue sample containing as few as 20 cells can be sufficient |
| How is STR analysis so accurate? | While the forensic scientists only test a few selected portions of DNA--usually 13 STR markers, the probability that two people (who are not identical twins) would have the same set of STR markers is vanishingly small. |
| The Innocence Project | A nonprofit organization dedicated to overturning wrongful convictions, uses STR analysis of archived samples from crime scenes to revisit old cases. As of 2010 more than 250 innocent people had been released from prison. |
| Genetic profiles used to identify casualties in after the World Trade Center attack | More than 10,000 victim's remains were compared with DNA samples from personal items, such as toothbrushes, provided by families. Ultimately, almost 3,000 victims were identified this way. |
| Likelihood of two people having the exact same 13 STR markers | Somewhere between 10 billion and several trillion |
| In what way are plants easier to genetically engineer than most animals? | A single tissue cell grown in culture can give rise to an adult plant. Thus genetic manipulations can be performed on an ordinary somatic cell then be used to generate an organism with new traits |
| The most commonly used vector for introducing new genes into plant cells | A plasmid called the Ti plasmid |
| Ti plasmid | A plasmid of a tumor-inducing bacterium (the plant pathogen Agrobacterium) that integrates a segment of its DNA (T DNA) into a chromosome of a host plant. The Ti plasmid is frequently used as a vector for genetic engineering in plants. |
| Example of genetic engineering in plants in India | In India, the insertion of a salinity resistance gene from a coastal mangrove plant into the genomes of several rice varieties has resulted in rice plants that can grow in water three times as salty as seawater. |
| Public concerns about possible hazards with genetic engineering | Today, most public concern is not on recombinant microbes (e.g. viruses/bacteria) but on genetically modified (GM) organisms used as food. |
| GM organism | An organism that has acquired one or more genes by artificial means; also known as a transgenic organism. E.g. genetically modified corn/soybean/canola crops. |
| Concerns with GM crops: environment | Some fear GM crops will pass their new genes to close relatives in nearby areas. These herbicide, disease, and pest-resistant plants then could hypothetically become "super weeds" that are difficult to control. |
| Concerns with GM crops: health | Some people fear that the protein products of transgenes might lead to allergic reactions. However, proponents of GM crops claim that these proteins could be tested in advance. |
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