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HUSOP2014
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The main purpose of cloning DNA is?
To make multiple copies of a unique gene sequence
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T/F A biological approach to cloning DNA is much less effective than chemical means.
FALSE
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All of traditional DNA cloning approaches use __________ DNA structures.
self-replicating
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What are the two main classes of vectors?
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Name two functions of cloning vectors.
- Modify DNA sequences within a unique segment of DNA
- make multiple DNA or RNA copies of a specific DNA sequence
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What are expression vectors used for?
To synthesize protein coded by a cloned DNA sequence.
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Name several types of cloning vectors and comment on the amount of DNA they can hold.
- Plasmid (up to 5.0 kB insert),
- bacteriophage (up to 25 kB insert),
- phagemid (up to 25 kB insert),
- cosmid (up to 45 kB insert),
- YAC (yeast artificial chromosome)(100-200 kB insert)
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1 kB is how many bases? What is a kBp?
- 1000 bases,
- 1000 base pairs
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The most frequently used cloning vector is the __________.
Plasmid
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T/F Plasmids have double stranded, closed circular DNA with selectable markers.
TRUE
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The genes encoded by bacterial plasmids include those that __________.
Impart drug resistance
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pBR322 is a type of __________ vector.
Plasmid
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In regards to a pBR322, what do the outer and inner cirlces represent?
- outer represents where restriction nucleases cut the DNA
- inner circle denotes where the resistance genes are located.
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T/F A different restriction nuclease is needed to open a plasmid that is used on the DNA to be cloned.
FALSE
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What enzyme is used to seal cut plasmids and to add covalent bonds?
DNA ligase
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T/F Bacteriophages are double stranded DNA vectors.
FALSE
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Bacteriophages infect which particular type of bacteria?
E. coli
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Bacteriophages contain the selectable marker __________ .
Beta-galactosidase
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Transfected E. coli will stain blue when incubated in the presence of what galactosidase?
x-gal, 5-bromo-4-chloro-3-indoyl-?-D-galactosidase
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A phagemid can be propogated by either a __________ or a __________.
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Propagating a phagemid as a bacteriophage requires what?
Co-infection with a bacteriophage and a phagemid
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Cosmids are manipulated as __________, but are expressed as __________ in the cell.
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YAC's are propagated in which type of yeast?
Saccharomyces cerevisiae
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How is cloning possible with YAC's?
It is recognized as part of its normal complement of chromosomes.
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YAC's contain all of the following except:
A) selectable marker
B) centromere
C) telomeric DNA
D) Beta-galactosidase
B-Galactosidase
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__________ is an endonuclease commonly found in YAC cloning vectors. What does this enzyme do?
EcoR1- It creates sticky ends with 5' overhangs
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What is the difference between complementary and genomic DNA?
- cDNA only contains only the protein coding region of a gene.
- Genomic DNA contains the entire gene sequence.
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Besides the protein coding region, genomic DNA has what 4 other parts?
- Promoters,
- introns,
- exons
- 3' non-translated domain
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The key to cloning in eukaryotic cells is __________.
Glycosylation
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__________ can accept and express proteins in DNA within different constructs.
Cos cells
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What vector is used to express glycosylated proteins in insects?
Baculovirus
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T/F Baculovirus can enter mammalian cells and replicate.
FALSE
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Which type of vector is known as a shuttle vector?
Bacmid
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