Recombinant DNA
True or false: Vectors can have different levels of expression, including constitutive, where their expression is always on, inducible, where an external stimulus triggers its expression, or tissue-specific, where its expression is limited to certain tissues.
True
True or false: Homologous recombination (HR) involves the exchange of DNA segments between regions that are exactly homologous, allowing for accurate homology search by the single-stranded overhangs.
False; HR can tolerate small mismatches
True or false: Direct repair requires cutting out and replacing single nucleotides.
False - direct repair reverses lesions without removing bases
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EVIL f
EVIL eae
Name 2 methods of introducing recombinant DNA into cells.
Possible answers: heat shock, electroporation, liposomes, accelerated particles
Explain what a Holliday junction is.
Intermediate structure in DNA recombination where two homologous dsDNA molecules are joined, crossing over into each other.
Name two general types of DNA damage or lesions.
Possible answers: single-strand breaks, double-strand breaks, chemical damage to bases, intra cross-linking, inter-cross linking
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EVIL lajeigelijfs lskjg helijgs
EVIL aela
Name 3 ways restriction enzymes can differ from one another.
Possible answers: Recognition sequence, cut pattern (blunt or sticky), type (I, II, etc.), sensitivity to DNA methylation
Explain the role of Rad51, and state what type of organism it exists in (bacterial or eukaryotic).
Rad51 acts as the central recombinase and catalyzes strand invasion in eukaryotic cells by forming filament on the invading strand.
Briefly describe what is involved in the NER pathway and how it differs from BER.
Nucleotide excision repair (NER) involves recognizing a lesion, opening the DNA around it, cutting out a short oligo containing the damage, resynthesizing the DNA and ligating it. NER removes a much longer stretch than BER.
Question
Answer
EVIL a
EVIL b
What are YACs, and how do they differ from BACs?
Yeast artificial chromosomes (YAC) are a type of vector used to carry large inserts. Unlike BACs, they can carry longer lengths of DNA and have all structural elements of a eukaryotic chromosome including telomeres, centromeres, and replication origins.
Explain the general role of the Ruv proteins in bacterial DNA recombination.
The Ruv proteins process and resolve Holliday junctions during recombination, driving branch migration and cleaving the junction so the DNA molecules can be separated.
Oxidative deamination is a type of DNA damage that can result in the conversion of C to U. Which form of DNA repair can be used to correct this?
Base excision repair (BER)
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nobody likes dna recombination
EVIL eljgir
EVIL efwji4 f4te
Describe the two “insertions” that need to occur for a successful transformation and explain why neither of them are 100% efficient (i.e., why the “insertions” may fail).
Gene needs to be inserted in the vector. The vector may simply re-ligate without taking up the insert.
The vector needs to be taken up by the cells. Cells do not readily take up foreign DNA, and many factors such as cell health, membrane permeability, etc. can affect whether the cell takes up the plasmid, maintains it, and expresses it.
List the following steps of homologous recombination in the correct order:
A. Double strand break
B. DNA synthesis and formation of Holliday junctions
C. End resection and formation of 3’ overhangs
D. Strand invasion and formation of a D-loop
A, C, D, B
Name and describe the two major pathways to correct double-strand breaks.
Homologous recombination - dependent on a sister chromatid (homologous DNA), only possible during S or G2 phase
Non-homologous end joining (NHEJ) - random rejoining, error-prone
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EVIL 2gsjlr
EVIL wji