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78 Cards in this Set
- Front
- Back
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What is recombinant DNA?
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When a scientist takes two segments of DNA (often from two different species) and combines them in vitro (in a test tube).
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The manipulation of of organisms or their components to make useful products is called...
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Biotechnology
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The direct manipulation of genes for practical purposes is called...
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Genetic engineering
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What do microarrays do?
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They are pictures of 2,400 human genes and their relative expression levels are expressed by colors, and can be useful in comparing perhaps the genes of a cancerous cell and that of a non cancerous cell to see which gene is harmful and being expressed more.
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Why is studying a eukaryotic gene a challenge?
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Because on one strand of DNA there are several genes (perhaps 1/100,000 of a strand constitutes a gene), only a small part of a gene actually codes for mRNA while the rest are noncoding sequences, and it there are no other indicators as to where a gene stops and starts other than a few promoting sequences.
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When scientists prepare copies of well-defined segments of DNA, it is called...
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DNA cloning
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What do bacteria have that make them useful for DNA cloning?
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They have plasmids, which are small circular DNA molecules that replicate separately from the regular circular bacterial chromosome.
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How do researchers clone the DNA in a bacterium?
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They take the plasmid from inside and combine a gene with it, forming recombinant DNA, and inject it back into the bacteria, forming a recombinant bacterium. The cell then divides producing exact replicas of itself, performing gene cloning.
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What are the two basic purposes for which gene cloning is useful, and give some examples.
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It is useful for making several copies of a particular gene (such as a gene that one plant has for immunity, but can be copied and injected into another plant for its own immunity), and is useful for making many proteins from the combined gene, such as the gene that makes the protein human growth hormone.
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Enzymes that cut DNA molecules at a limited number of specific locations, such as the ones that protect bacteria by cutting up the viral DNA that infects it, are called...
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Restriction enzymes
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Each restriction enzymes is very specific, recognizing a particular short DNA sequence at a particular site and cutting both DNA strands at different points within this site. What is this site called?
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The restriction site
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What protects the bacteria's own DNA from being but by restriction enzymes?
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The addition of methyl groups to the A and C of the DNA.
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Discuss the formation of restriction fragments.
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Restriction enzymes usually recognize 4-8 nucleotides (which are symmetrical, meaning they are identical when each is read in the 5' to 3' direction), and cuts them into restriction fragments, which can occur many times along one set of DNA, producing many fragments.
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What are sticky ends, what do they do, and what can they ultimately form?
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When the restriction enzyme cuts the DNA at different places along the identical DNA strands, then a long, unpaired end is left sticking out and can form hydrogen bonds with other restriction fragments cut by the same restriction enzyme. DNA ligase can make these new hydrogen bonds permanent, forming recombinant DNA.
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T/F: One the restriction enzymes cut DNA strands at the restriction site and form sticky ends, the cut DNA can form hydrogen bonds again with itself.
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True
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A DNA molecule that can carry foreign DNA into a host cell and replicate there, such as the plasmids of bacteria, are called...
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Cloning vectors (bacteria replicate the fastest so they are the best cloning vectors.
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After reviewing the list of procedures of cloning genes in bacterial plasmids through the shotgun approach, and knowing that all the alive white bacteria contain DNA from the entire bird's genome, what is all this remaining bacteria called?
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A genomic library
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T/F: Certain bacteriophages have also been used as cloning vectors for making genomic libraries.
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True
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Why are bacteriophages good cloning vectors?
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Bc once foreign DNA is cut up with a restriction enzyme, it can be shortened a bit and put into the phage to be injected into a bacteria and reproduces many times again, but it carries many more base pairs of foreign DNA than does a plasmid.
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What are Bacterial Artificial Chromosomes and how are they used as cloning vectors?
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They are just larger plasmids which are cut down to only contain the base pairs necessary for replication, and can hold a much larger insertion of foreign DNA, making less clones/books necessary to complete the library. They are also organized nicely into the little plastic compartments.
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Describe the formation of complimentary DNA (cDNA) in eukaryotic cells.
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Reverse transcriptase obtained from retroviruses is used to turn mRNA into a single strand of DNA and uses the poly A tail to create a many-Thymine primer. Then, DNA poly II makes the second strand of the DNA to form one complementary DNA strand.
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What is different about a cDNA library?
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Many mRNA molecules are extracted from a cell and are turned into cDNA by the long process described before. With the addition of restriction sites on each end, the cDNA can be added to a vector, and many different mRNA strands into cDNA strands are transformed into the vector making a cDNA library with different cDNAs incorportated into different vectors, of course. However, this isn't a representation of the entire genome, bc only the mRNA strands transcribed from the original cell were expressed in the library, not the entire genome.
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If you wanted to study the pattern of gene expression in cells or wanting only to find the coding sequence of a gene, which library would be best?
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A cDNA library, which takes out all the introns and exons an shows which mRNA/genes are expressed.
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If you wanted to study introns and exons in a gene or you want to clone a gene of an animal but don't know which cell type expresses it, which library would be best to use?
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A genomic library
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Now that we have the entire genomic library of the hummingbird in the bacteria vectors, what can we make to find the beta-globin gene we are looking for?
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Knowing at least part of the gene's sequence (either from the amino acids in the protein or knowing the sequence in a similar species) we can make a nucleic acid probe, which is a single strand of DNA or RNA that is complimentary to part of the gene and is marked with radioactive isotopes.
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What is nucleic acid hybridization?
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It is when you take the fluorescent nucleic acid probe and perform nucleic acid hybridization, in which the complementary DNA of the gene will bind to the nucleic acid probe to mark the gene's existence.
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How does the nylon membrane plate work in nucleic acid hybridization?
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Each clone in a well of the multiwell plates is place on one place of the multinylon membrane, which is designed to denature the bacteria and make all its DNA single-stranded. The nylon membrane is then immersed in a solution with the nucleic acid probes and those that show radioactivity have bound to the probe and indicate the gene of interest.
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T/F: Once the bacteria containing the gene of interest is found the gene can be copied and used in prokaryotes or eukaryotes for ultimate use, and using either has advantages and disadvantages.
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True
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What is an expression vector?
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It is a cloning vector such as a bacteria in which scientists will place a promoter sequence JUST upstream of the restriction site. Then, when restriction enzymes cut at the site and the foreign DNA is incorporated, the bacterial enzymes will recognize the promoter and express the foreign DNA and its products. Yay!
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Expressed cloned eukaryotic genes in prokaryotes poses the problem of introns and exons, bc introns can make a gene long and unnecessary. How is this overcome?
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By using the cDNA sequences which are derived from the mRNA, which has already had its introns spliced and sent away.
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Why are yeasts good options as eukaryotic cloning vectors?
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Because they produce just as quickly as bacteria and also contain plasmids, which is rare amongst eukaryotes.
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Why are using Yeast Artificial Chromosomes so helpful?
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Because they are actual chromosomes (having a centromere, origins of replication, and telomeres) and divide as a regular chromosome does during mitosis; and bc this chromosome is so much larger than a plasmid that the probability of an ENTIRE foreign gene being contained in it (rather than being spliced up amongst many plasmids) is much greater.
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Why might the use of host cells from an animal cell as cloning vectors be necessary?
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Because even if the plasmids or artificial chromosomes of yeast and bacteria can produce the intended product of a gene, they may not have the tools required to carry out the protein modification that occurs after translation, as animal cells have.
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What is one main way that scientists introduce foreign DNA into animal cells?
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They use electroporation, which uses a small electric pulse to open holes in the plasma membrane into which foreign DNA can be directly injected.
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What is the Polymerase Chain Reaction (PCR) used for?
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It can target and amplify (copy many times) a specific sequence of genes in vitro much quicker than DNA cloning, and can even be used to make several copies and to inject them into a vector rather than putting an entire genome into several vectors. The book says it's like photocopying "one page" of the book rather than checking out the whole library.
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What 4 things does PCR require?
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It requires DNA containing the target sequence, heat-resistant DNA polymerase (derived from bacteria that live in hot places), and two DNA strands complementary to each end of one strand of the DNA of interest to serve as primers.
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After 3 cycles of PCR and thus a yield 8 (2^3) DNA molecules of interest, how many will be an exact copy of the first?
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2 of the 8 (then grows to almost 99% being exact copies after about 30 cycles)
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T/F: For high specificty as PCR yields, how many nucleotides long must the primers be?
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About 15
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Why doesn't PCR replace cloning vectors?
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Because occasional errors in replication do occur, whereas in cloning vectors the actual original DNA is incorporated so no errors can occur (and after each replication enzymes in the bacteria proofread the replication)
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T/F: Using the polymer medium and the positive charge on the other end, gel electrophoresis brings all DNA strands toward the opposite end of the gel, but only the smaller ones travel very far. So, the medium separates the bands by charge and length.
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True
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How can gel electrophoresis be used in restriction fragment analysis?
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When one obtains all the restriction fragments cut up by resctriction enzymes in a cell, they can use gel electrophoresis to create the band pattern. Each band pattern shows the use of a particular restriction enzyme and DNA sequence, so viola! From the fragments we can find out the restriction enzyme used, AND what the DNA looked like before it was digested/cut up.
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How can gel electrophoresis distinguish certain alleles of a certain gene?
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Because if the different allele has a mutated base pair that is at a RESTRICTION SITE, such as in sickle-cell, then the restriction enzyme will not recognize it and the segments of the gene will be longer. Gel electrophoresis will show this mutated form of the gene.
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When replication of a single DNA strand occurs (when provided with regular nucleotides and dideoxyribonucleotides marked with color) and the replication stops at the ddNTPs and a length spectrum chart is created, what is this called and what does it do?
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It is called Dideoxy chain termination method, which can sequence an entire gene.
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Transcription levels are commonly used as a measure of gene expression. How can we measure them and find out when, and in which cells, genes are expressed?
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By using nucleic acid hybridization with mRNA molecules instead of genes to tell if an mRNA exists in that cell and when it exists in that cell.
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What is the difference in Northern blotting from Southern blotting?
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The same technique is used but with mRNA instead of a genome. (e.g., we can find out by taking the mRNA from hummingbirds in each life cycle when the beta-globin protein is being expressed).
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How is reverse transcriptase-polymerase chain reaction (RT-PCR) different from PCR?
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In this case, mRNA from the bird is used and converted to cDNA, and PCR occurs only with the primers that prime beta-globin genes. So, when put in gel electrophoresis, only those sample with many copies of the beta-globin gene were producing beta-globin at the time.
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What is it called when you used fluorescent probes to mark genes in a species without extracting anything from it?
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In situ hybridization, duh.
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How can the expression of genes be measured?
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By making the mRNA already present in the cell into cDNA and using nucleic acid hybridization (in a spliced/cut-up genome) to identify which gene corresponds to the mRNA and thus which gene is being expressed at a certain time.
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If you are given a tissue sample and a microarray (plate) of the organism's entire genome, what can you do to find out which genes are currently being expressed in those cells?
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Isolate the mRNA, turn it into cDNA and label with fluorescence, and place these into all the holes of the microarray containing different genes. Those that hybridize and become fluorescent are the genes that are currently being expressed.
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What is the procedure on the last slide called?
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DNA Microarray Assay of Gene expression levels
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What is invitro mutagenesis?
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When a mutated form of a gene is created and then placed into an organism so that the healthy cells with normal genes are knocked out. The subsequent phenotype of the cell as a result of the mutated gene can basically be used to determine what that gene is supposed to do!
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What is RNA interference? (RNAi)
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When small double-stranded RNA is used to stop the translation of complementary mRNA strands in order to determine the function of this mRNA/gene (which will be determined by the phenotype in its absence)
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What is one of the main, emerging functions of thins that stop the translation of genes like RNAi?
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To ultimately stop one gene at a time in an organism from being expressed, so that the relationship between groups of genes can be analyzed (the ultimate theme of biology).
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T/F: Organismal cloning is neither gene cloning from bacteria nor cell cloning from asexual reproduction, but is rather a the creation of a genetically identical organism from one cell of an existing organism.
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True
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Why is there in interest in cloning other than it's awesome?
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Because from clones we can generate stem cells which can generate many different tissues.
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What is a totipotent cell?
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A somatic cell, like that of a carrot, that can be extracted and put into a culture medium to grow into another fully grown carrot genetically identical to the parent plant. Basically, the specified carrot cell can dedifferentiate and make all the necessary cells for a carrot, so it's a totipotent cell.
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What is nuclear transplantation?
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When you take out the nucleus of an egg and replace it with the nucleus of a differentiated cell to see if full genetic capability is maintained. (It was determined that something in the nucleus DOES change as animal cells differentiate).
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Describe the first organismal cloning of a mammal.
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When mammary cells from sheep #1 were starved and dedifferentiated, they were then placed with an enucleated egg from sheep #2. When the dedifferentiated mammary cells fused with the egg lacking a nucleus, and were implanted in random sheep #3, the offspring was genetically identical to sheep #1 who gave the mammary cell. So this nucleus gave rise to a clone.
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What has cloning of animals ultimately taught us?
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That environmental factors play a big role in development, so even clones will look different, and that clones often have serious defects.
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Why does the dedifferentiation of differentiated cells and using them for donor nuclei often pose problems?
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Bc after the cells have differentiated, some of their genes have been turned off by methylation and are not reversed before being utilized as an embryonic nuclei, causing problems.
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T/F: There are very non-specific, self-reproducing stem cells in the human embryo, but there are also stem cells in the adult human that exist to replace differentiated cells that do not divide and replace themselves, such as blood cells.
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True
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If a stem cell is pluripotent, it is...
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Capable of differentiating into many, many different cell types
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When the main aim of cloning is to produce embryonic stem cells to treat disease, it is called...
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Therapeutic cloning
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What are induced pluripotent stem cells (iPS cells) and why are they important?
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They are regular cells (like a skin cell) that are turned back into embryonic stem cells and are important because they do not involve full formation of an embryo and stem the ethical issues.
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Variations in a DNA sequence (such as those that are responsible for alleles) are called...
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Polymorphisms
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When a single base pair mutation occurs about every 100 to 300 base pairs either in the coding or non coding sequence of DNA it is called...
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A single nucleotide polymorphism (SNP)
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What is an example of a restriction-fragment length polymorphism and what is it?
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Sickle cell disease, where the single nucleotide polymorphism (SNP) is at the restriction site and so the restriction fragment length is screwed up.
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How can RFLP and SNP help us diagnose genetic disorders?
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Because one type of SNP is common among a certain type of disease in a family. If one person has the SNP (mutation), and if it is close enough to a gene, then during crossing over the mutation and gene are likely to stay together and be crossed to offspring.
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Introducing genes into an afflicted individual for therapeutic purposes is...
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Gene therapy
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How can a retroviral vector be used in gene therapy say of bone marrow and blood cells?
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If a normall allele of the defective allele is obtained and made into complementary RNA and inserted into a viral cloning vector, extracted cells from the person's bone marrow can be infected with the altered viral vector which will change the DNA. These newly altered bone marrow cells are then placed back into the person's bone marrow.
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T/F: Vaccines can be either the proteins that are on a pathogen that trigger the immune response, or weakened forms of the actual pathogen itself that trigger the immune response.
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True
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What is a transgenic animal?
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An animal that was fertilized by egg and the sperm of its own kind, but had a gene introduced into its embryo from a third source, so it expressed that gene, such as the fluorescent pig! (so human proteins are produced in transgenic farm animals)
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A person's genetic profile is basically their...
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Fingerprints, etc.
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Simple sequence DNA containing multiple tandemly repeated units of two to five nucleitodes that act as genetic markers for things like quick crime analysis, are called...
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Short tandem repeats (STRS)
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T/F: Microbes and their traits are currently being used and their genetics exploited for environmental clean up
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True
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In plants, what is the plasmid usually used for genetic engineering in plants that integrates a segment of its DNA into a chromsome of a host plant?
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A Ti plasmid
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T/F: Using a Ti plasmid, you can enter the recombinant plasmid literally into the cells of the plant itself or back into the bacterium and onto the surface of the plant to form a transgenic plant.
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True
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What is the main debate amongst food today in biotechnology?
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Genetically modified food
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