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18 Cards in this Set
- Front
- Back
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DNA cloning
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producing many pieces of DNA (copy) for manipulation
-produce human insulin in bacteria |
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DNA cloning step 1
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cut human genome into manageable pieces with restriction enzymes
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restriction enzymes
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enzyme that recognizes and cuts a specific DNA sequence (palindroms), makes staggered cuts
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staggered cuts
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leave sticky ends that can stick to DNA cut by the same restriction enzyme
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DNA cloning step 2
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insert pieces of human genome into bacterial vectors
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vectors
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plasmid- circular piece of DNA
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parts of a vector
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1. bacterial promoter
2. bacterial origin 3. selectable marker- allows us to identify which bacteria are carrying the vector- AmpR 4. multiple cloning site- contains lots of RE binding sites, wherer we insert foreign DNA |
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recombinant DNA
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cut genome and vector with same restriction enzymes, put together (matching sticky ends), seal nicks with DNA ligase
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DNA cloning step 3
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-put recombinant DNA into bacteria by transformation, when bacteria are stressed they take up DNA
-stress by heat or electric shock -plate bacteria on media (bacteria food) w/ antibiotic to select ones that took up recombinant DNA |
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library
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pieces of entire genome on bacteria
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DNA cloning step 4
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find insulin gene by DNA hybridization
-transfer bacterial colonies to a filter -denature DNA (change in heat, salt) -usea labeled, complementary probe |
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other techniques
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-polymerase chain reaction (PCR)
-reverse transcriptase PCR (RT-PCR) -agarose gel electrophoresis -dideoxy DNA sequencing |
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polymerase chain reaction (PCR)
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-makes many copies of a single gene: amplification
-certain requirements -30 cycles of 3 temps |
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PCR requirements
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1. DNA polymerase
2. template- genomic DNA 3. dNTP's (nucleotides) deoxyribonucleotide triphosphate 4. primers gene specific |
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30 cycles of 3 temps
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1. 95 degrees C- denaturation cycle
2. 45 degree C to 55 degrees C- annealing primers bind your gene 3. 68 egrees C to 70 degrees C- extension phase |
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RT-PCR
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-looks at mRNA -> gene expression
-viral enzyme that copies RNA to DNA -copy mRNAs into DNAs -> cDNA (complementary DNA) -amplify cDNA's (copies of mRNA) |
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cells w/ no drug
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lots of RT-PCR
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cells w/ drug
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no RT-PCR product
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