Cloning Process (Page 2) - Ap-bio.com

twoeggfinnishBiotechnology

Dec 14, 2012 (4 years and 8 months ago)

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Chapter 20

DNA

Technology & Genomics

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Biotechnology Terms


Biotechnology


Process of manipulating organisms or their components to
make useful products


Genetic engineering + tissue/cell culturing technologies


Genetic Engineering


Manipulation of individual genes or entire genomes


Insulin (insulin


E. coli bacteria OR yeast) & GMO
(Genetically Modified Organism)


Recombinant DNA


Artificially created DNA


Typically, DNA is integrated from another species


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Biotechnology Terms (Page 2)


Gene Cloning


Laboratory production of multiple copies of DNA segment


Therapeutic cloning


embryonic stem cells


Spinal cord injuries


Reproductive (
organismal
) cloning


Dolly the sheep


Restriction Enzymes


Enzymes that cut DNA at specific locations


Usually, derived from bacteria


Cut sites of DNA =
restriction fragments


Sticky ends


restriction fragments usually have one end
longer than the other


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Quick Assignment


Relate the 6 terms just discussed in a concept map.


Be prepared to defend your arrangement

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Cloning Process


5 steps (first 2)

1. Identify & isolate the gene of interest


Involves finding a
cloning vector



plasmid or organism used
to carry the DNA sequence to be cloned


2. Cut gene of interest from original site & open up vector’s
DNA using a ________ ________


This ensures matching sticky ends on gene of interest &
vector DNA


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Cloning Process (Page 2)


5 steps (3
-
4)

3. Combine the 2 DNA pieces (into a recombinant plasmid?)


Recombinant plasmid



plasmid + DNA fragments


Sealed together using
DNA
Ligase


Remember: we used ________ ________ to cut gene of
interest from original site & cut vector’s DNA


This ensures matching sticky ends on gene of interest &
vector DNA


4. Transfer the vector (recombinant plasmid) into a host cell


Usually involves
bacterial transformation



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Bacteria & Genetic Recombination


Conjugation


Bacterial Sex


Genetic material is exchanged
by direct contact



Transduction


Phage transfer of DNA


Involves a phage vector


Phage moves the DNA from
bacterium to other bacterium

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Bacteria & Genetic Recombination


Transformation


Uptake of exogenous
DNA


Griffith’s experiment
-

pathogenic DNA was
transferred to benign
bacteria


Most common method
for genetic engineering


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Step 5


Select for transformed cells


Link the gene of interest with
a reporter gene


Such as
pBLU

or
pGLO


pBLU

= Blue coloration


pGLO

= fluorescent green
under UV light



In Lab 6, we will insert the
coloration gene and an
ampicillin resistance gene to
select for transformed cells


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At this point…


You know which cells have the gene of interest


You can identify the cells that have the gene of interest


Now what?


You need to extract the gene of interest


How would you do that?

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Nucleic Acid Hybridization


Detects the gene of interest


Uses a short, single stranded DNA or RNA called a
nucleic acid probe


The nucleic acid probe is complementary to a known
sequence in the gene of interest


Usually attach a radioactive isotope or fluorescent tag
protein so that it is detectable

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Genomic Libraries


Nucleic Acid Hybridization repeated many times
produces a
genomic library


Thousands of recombinant clones


Each has a piece of the original genome being studied



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cDNA

Library


cDNA

= complementary DNA


mRNA is extracted from cells


Use what enzyme to make DNA from this mRNA?


Then make another strand of DNA using what enzyme?


cDNA

library is only a portion of the genome


Portion that codes for mRNA


Exons
? Introns? tRNA?
rRNA
?

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Microarray Assay


Genome
-
wide study of gene expression


Different genes are in each well


Identifies gene interactions + provides clues to gene
functions


Take samples throughout development + assay to
determine which genes are expressed and at what
stages


Detect patterns of expression throughout development


Detect likely response to a pathogenic agent


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PCR




Polymerase Chain Reaction



Thermal cycling



Amplification of DNA



3 Steps



Denaturation (Heating)




Annealing (Cooling)


Primer formation




Extension



DNA polymerase adds
nucleotides at 3’ end


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Gel Electrophoresis



DNA is negatively charged so it
moves AWAY from the (
-
) cathode
toward the (+) anode

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Southern Blotting


Used to detect specific DNA sequences


Useful for comparing samples


Combines gel electrophoresis + nucleic acid
hybridization


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DNA Technology affects us…


Disease Diagnosis


PCR used to detect traces of viral DNA or RNA in sample


RFLP (Restriction Fragment Length Polymorphisms)


Different alleles have different
RFLPs


Gene Therapy


alter afflicted genes


Pharmaceutical Production


Insulin production


Forensic Application


DNA fingerprints


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Page 2


Environmental cleanup


Genetically engineered microbes


Detoxification of specific wastes


Agricultural applications


Insert pest
-
resistant or drought
-
resistant genes


GMO (Genetically Modified Organisms)


You eat GMO corn, soybeans, canola and cottonseed oil


Probably at least weekly


46% of
GMOs

are grown in US


Europe had 12 year moratorium on growing GE foods