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18 Cards in this Set
- Front
- Back
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what is the definition of recombinant DNA
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joining DNA molecules, usually from different biological sources, to form a DNA molecule that is not found in nature
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what are the 3 basic steps in DNA cloning with vectors
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1. cut DNA and vector with similar restriction enzymes
2. incorporate DNA into vector and connect them with DNA ligase 3. introduce vector to host cell and replicate cloned DNA |
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what are restriction enzymes
-what are the sequences they recognize called -how are they joined |
restriction endonucleases that identify specific recognition sequences (restriction sites) and cleave DNA into fragments and produce "sticky ends" and sometime overhangs
-palindromic (AACGTT) -DNA ligase |
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definition of a vector
-what are the 3 qualities a vector should have |
carrier DNA molecule that replicates the target DNA fragment
-replicate independently (isolate) from host cell -selectable gene marker (amp) -several restriction sites |
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what is the most typical vector used
-how is it incorporated into host cell (2 techniques) |
plasmid (extrachromosomal dsDNA)
-transformation (1. Ca2+ ions with brief heat shock; 2. electroporation, pulse of electricity) |
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what are expression vectors
-can be used in what types of cells -what are typical host cells |
allow for the expression of the cloned gene (introduce promoter)
-prokaryotic, eukaryotic -plant, animal, bacterial, yeast (most popular for eukaryotic cells) |
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what does PCR eliminate the need for
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host cells
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what are the 4 requirements for PCR
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DNA polymerase
Mg2+ 4 dNTPs 2 primers |
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what are the 3 steps for PCR
-result |
1. denaturation (highest T)
2. annealing (lowest T) 3. elongation (middle T) -doubles DNA each cyle |
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what are the 3 problems with PCR
-major application for PCR |
some info about target DNA must be known to make primer, contamination, cannot amplify long segments
-can amplify fragments of a gene in order to easier analyze and see its functions |
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what is gel electrophoresis
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separation of DNA molecules with respect to size
-small DNA most faster -all DNA move towards + end because they are all negatively charged |
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what is GFP, why is it useful
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glows green
-provides mechanism to visualize gene expression of our cloned, or target DNA |
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what are DNA libraries
-2 types of DNA libraries |
collection of cloned DNA samples from a single source (tissue, cell individual)
-genomic library -cDNA library |
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describe genomic library
-how is it made |
contains at least one copy of all sequences of the genome
-DNA genome is cut with restriction enzymes, ligated into vectors |
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describe cDNA library
-how is it made (2) |
mRNA is reverse transcribed into DNA, showing all the active genes at that time
-isolate mRNA, reverse transcriptase to get cDNA, clone cDNA in vectors -3' RACE: ss cDNA from mRNA, PCR to amplify all the active genes |
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what are microarrays
-why are they important |
collection of ssDNA attached to a chip
-allows thousands of genes to be studied simultaneously |
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how is hybridization and detection used in microarrays
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complementary strands can be introduced and hybridize with ssDNA on microarray to visulaize which genes are expressed in the genome
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what are required for Sanger method
-how does the Sanger method work |
primer, DNA poly, template, ddNTPs, detection system (gel)
-when ddNTP is incorporated into the growing strand with DNA poly, synthesis is terminated. The sequences are plated on gel electrophoresis, smallest strands fall to bottom. Read from bottom up is 5' to 3' of the strand complementary to the template/target sequence |
