Term
| What is so useful about restriction enzymes? |
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Definition
| A given enzyme will always cut a given DNA molecule at the same sites |
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Term
| When enzymes cut at a longer sequence of nucleotides, they cut (more/less) frequently |
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Definition
| Less frequently because that long sequence is less likely to happen |
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Term
| Why are they called "restriction" enzymes? |
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Definition
| Because they restrict transformation in bacteria. Restriction means methylation |
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Term
| What are the two basic types of restriction enzymes? |
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Definition
| One cuts blunt edges, the other cuts overlapping edges |
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Term
| Which are more valuable, blunt edges or overlapping ones? Why? |
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Definition
| Overlapping edges can easily be inserted with a new nucleotide chain |
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Term
| What are two ways to analyze the cut up pieces of DNA? |
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Definition
| Agarose gel was invented, can tag the DNA with radioisotopes and move the gel to photopaper, OR stain with ethidium bromide which does not bind |
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Term
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Definition
| Making a small segment of complementary DNA and tagging it to identify the DNA you are looking for |
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Term
| What does hybridization depend on? |
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Definition
| The fact the complementary DNA sequences can be denatured (by heat) and upon cooling they return to the original form |
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Term
| What is Southern Blotting? |
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Definition
| Take a run of DNA on a gel, put it onto a wet sponge, add nitrocellulose and paper towels which pull the gel. Adding a DNA probe with the blot inside a plastic baggy allows the probe to bind to the target DNA |
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Term
| How does methylation of DNA affect restriction enzymes? |
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Definition
| Methyl groups on methylated DNA stop restriction enzymes and keep DNA intact. Unmethylated DNA can be cut easier |
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Term
| What first compared the DNA of chimpanzees and humans? |
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Definition
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Term
| Describe the enzymatic method of DNA sequencing |
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Definition
| Take double stranded DNA, add a labeled primer (single-stranded), add dideoxyribonucleotides (ddNTPs), add DNA polymerase. cuts are made at each ddNTP, and you run the fragments on a gel to determine the sequence |
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Term
| What is the shotgun strategy? When does it work? |
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Definition
| Assembling a genome after random reads from enzymatic sequencing. Because of so much excess genetic material, shotgun method is limited to simple organisms lacking lots of introns |
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Term
| How did restriction and other enzymes make cloning possible? |
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Definition
DNA Polymerase III -> DNA elongation
Ligase sealed gaps (joined 2 together) |
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Term
| What is Berg's A/T Tailing? |
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Definition
| A plasmid containing an A/T section receives a piece of viral DNA |
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Term
| How did the Boyer-Cohen Experiment verify cloning? |
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Definition
| They joined a blunt end with a staggered end; tried to add a tetracycline resistant gene. The experiment added a different enzyme, which was reproduced. Insertion was accomplished with DNA ligase. They actually patented the process |
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Term
| What is the action of DNA ligase? |
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Definition
| It provides the energy to secure the circle of a plasmid |
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Term
| How may a plasmid be inserted into bacteria? |
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Definition
| Using Ca++ or electric shock |
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Term
| What are the 3 required components for a vector? |
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Definition
| origin, region of genetic interest (like tet resistance) and a cutting spot |
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Term
| Cloning something into a plasmid which covers the area of tetracycline resistance does what? |
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Definition
| removes the resistance to tetracycline |
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Term
| How is the lac-Z plasmid used? |
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Definition
| use the lac gene to change a molecule that gives a color. the check cloning at that gene, they use a color reaction. |
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Term
| What is the isolation of Poly(A) RNA? |
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Definition
| a solution of cellulose with oligothymidines attached, run RNA thru that, and the Poly-A tails of mRNA bind and stick in the cellulose. Can remove pure mRNA this way. |
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Term
| What is a second use of Poly-A RNA besides isolating mRNA? |
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Definition
| The mRNA can be used with a TTTT primer and reverse transcriptase to synthesize cDNA (copy DNA). Then wash off RNA and synthesize the other DNA strand. This DNA has NO INTRONS b/c it has only CODING DATA and is thus different from genes |
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Term
| How does hybridization test for sickle-cell? |
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Definition
| a plasmid and mRNA binds, and can locate specific DNA. Sickle cell has a Val instead of a Gly. Probe for both normal genes and sickle cell probes. |
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Term
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Definition
| Amplifies DNA. DNA+heat+hybridization of oligonucleotide primers-> ddNTPs, polymerase, and then DNA synthesis. There's a cooling and the primers anneal. A heat cycle is required, and new polymerase is required for each cycle |
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Term
| What are DNA micro arrays? |
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Definition
| glass microscope slides studded with a large number of DNA fragments, each containing a probe for a specific gene. This monitors the expression of thousands of genes at once |
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Term
| What does the term "transformation" mean to a a bacterial cell? |
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Definition
| The cell uptakes DNA from its surroundings and incorporates it into the genome by recombination. This transforms the bacterial strain into another |
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