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26 Cards in this Set
- Front
- Back
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What are recombinant DNA? |
genes or DNA from two different sources combined 'in vitro' (outside of the organism) into the same molecule. |
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What are two sections of a typical gene? |
1. Promotor - Controls transcription of mRNA and is host specific. 2. Coding regions (exons, introns cut off) |
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What is biotechnology? |
The manipulation of organisms or their components to make useful products. |
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What is genetic engineering? |
The in vitro (outside the organism) alteration or recombination of genetic material and putting it into a living organism. |
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Which is more beneficial between genetic engineering and selective breeding? |
Genetic engineering because: - It can control gene expression - Genes can be introduced from any species - Precise choice of genes However traditional methods are used for ethical reasons. |
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In genetic engineering, how is the gene of interest prepared? |
It must be cloned. This involves joining sequences from different sources to form the DNA sequence. |
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What are cloning vectors? |
DNA molecules that can carry foreign DNA. eg Plasmids, bacteriophages |
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What are plasmids? |
Circular autonomously replicating DNA in host bacterial cell. Used in GE because it can be easily isolated from the bacterial host and easy to insert DNA in vitro and reintroduce to the host. |
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What host bacteria is commonly used in GE? |
E coli , which is found in the small intestine of warm blooded animals. Used because it is easily grown and DNA can be introduced into the cell. |
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What is a competent cell? |
A cell that readily takes up DNA (by transformation) Bacterial cells are made competent by being treated so they can take up plasmid DNA. |
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How do selective markers work? |
There are two types, 1. To make sure the cell has taken up a plasmid. (by antibiotic resistance) 2. To make sure the plasmid has taken up the foregin gene. (by colour) eg X-GAL (artificial) is Galactose-indigo (clear) and the 'functional' plasmid (no foreign DNA) produces an enzyme that breaks this up into Galactose + blue pigment (so blue bacteria). Plasmid with foreign DNA does not produce this enzyme and hence the X-GAL cannot produce blue pigment, so the clear bacteria are the bacteria carrying the plasmid AND the foreign gene. |
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What is a genomic library? |
Collection of clones that together contain an entire genome of an organism. |
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What are BACs? |
Bacterial artificial chromosomes which can hold many inserted DNA fragments and it works similar to plasmids in bacteria. |
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Compare plasmid libraries and BAC libraries. |
Plasmid libraries can only insert small fragments of DNA into each plasmids, so needs many copies. BAC libraries can insert huge fragments of DNA into its chromosome, so less clones are needed. this one is stored as DNA or cells in a multi well plate. |
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What is a cDNA library? |
Collection of clones that contains all gene sequences expressed in a particular tissue type. It is made from mRNA molecules. less complex as genome libraries as no promoters, untranslated regions or introns. |
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How is cDNA prepared? |
c = complementary mRNA -> cDNA The mRNA in the cytoplasm of a cell is extracted. The enzyme reverse transcriptase (from retroviruses) is used to make a complementary DNA strand. DNA polymerase then uses that as a template strand to make the complementary DNA strand, forming cDNA. |
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You want to clone a gene, but unsure of what tissue type it is expressed in, which library would you use? |
Genomic library |
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You are interested in the regulation of the gene, which library would you use? |
Genomic library (regulation involves promoters, cDNA libraries do not have this) |
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You are interested in the coding sequences of a gene, which library would you use? |
cDNA |
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What is DNA hybridization? |
It allows us to sort through thousands of clones in a library to find a gene of interest by base pairing. |
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What is a DNA probe? |
A single stranded labelled DNA fragments with similar sequence as the gene of interest. It is tagged with radioactive or fluorescent label. |
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What is PCR? |
Polymerase chain reaction. It is a three step process that produces millions of copies of a targeted region of DNA. Quickly amplifies any piece of DNA without using bacterial cells. |
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What are the three things required in PCR? |
1. Heat stable DNA polymerase 2. Deoxyribonucleotides (dNTPs) 3. Two primers (1 on each strand) Why primer? Because DNA polymerase can only extend existing double strand regions. |
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What are the steps of PCR? |
1. Denaturation (heating to break DNA strand around 90 degrees) 2. Annealing (rapid cooling and complementary binding of primers 50 degrees) 3. Extenstion (heated up slightly and DNA polymerase extends the strand from the primer, making a double stranded DNA around 70 degrees) |
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What is Gel electrophoresis? |
Uses a gel in an electrical field to separate macromoleciles (DNA or proteins) depending on their rate of movement in this field. It depends on size, electrical size and other properties of the macromolecules. DNA seperation depends mainly on size (of fragment). Longer fragment = migrates less along the gel. |
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What is southern blotting? |
Electrophoresis + DNA hybridization. why bother? electropherisis results in so many DNA fragments that they could not be distinguished. Hence specific fragments are identified by souuthern blotting, using labeled probes that hybridize to the DNA. |
