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Flashcards in Exam 3 Part 3 Deck (22)
1

genetic engineering

human manipulation of an organism's DNA

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genetic engineering methods

recombinant DNA tech

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recombinant DNA technology

transplating a gene from one organism to another
rDNA is created using molecular cloning
DNA of interest is inserted into a cloning vector

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what would the final clone be used for?

used the gene product
find what the gene product does
diagnosis of a disease
gene therapy
mutation
sequence

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strategy of genetic engineering

1. isolate DNA of interest from an organism
2. cut the DNA with a restriction enzyme
3. cut the cloning vector with the same restriction enzyme
4. ligate piece into a cling vector to make a recombinant DNA molecule
5. transform the recombinant DNA into a host (like e. coli) the host cell will make identical copies called clones

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restriction enzymes

produce sticky ends
naturally occuring

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genomic library preparation

DNA is extracted from the organism of interest
cut DNA into small pieces using restriction enzymes
ligate pieces into cloning vector
form library

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genomic library

collection of clones/plasmids in which each plasmid contains a different piece of DNA

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why have genomic libraries?

screen libaray to look for a specific sequence
diagnostics
squence by shot gun screens

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screening a genomic library

goal: find a DNA sequence in a pool of DNA
use a DNA probe
caveat

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DNA probe

detectable tag on a primer

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cDNA library prepartion

RNA is extracted from an organism of interest
RNA is reverse transcribed into cDNA
Each cDNA is cloned into vector
Each library clone has a different cDNA molecule

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why have cDNA libraries?

Detect sequence of interest
tell difference bt exons and introns
splice varients

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splice variants

can tell where exons/introns are-coding regions

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uses for PCR

amplify a DNA segment of interest
forensics
organ matching
sequence an entire genome
quantify a transcript
make mutations

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Real-time PCT

quatitative PCR
measure the increase in the amount of PCR product
measure the abundanence of a transcript in a sample
measure cDNA as it is amplified

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PCR to inroduct mutations

study mutation
identify a disease
identify the important amino acid in a polypeptide
to understand the role of a specific amino acid in a polypeptide

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types of mutagenesis

site-directed mutagenesis
random mutatgenesis

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site directed mutagenesis

mutate one specific nucleotide
include primer with single nt change

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random mutagensis

create random mutatiosn ian DNA of interest
add Mn2+ instead of Mg2+
causes polymerase to have reduced fidelity

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Southern blotting

detect a specific DNA sequence in a sample
used to compare homologous genes
analyze intron organization
paternity tests
diagnostics
methylated sequences

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Southern blotting procedure

1. isolate DNA from organism of interest
2. cut into small pieces using restriction enzymes
3. sparate DNA fragments using gel electrophoresis
4. transfer DNA from gel to a membrane/blot
5. incubate blot with a labelled probe that is complementary to the DNA of interest
6. visualize the probe--band will be DNA of interest