Chapter 20: Recombinant DNA Technology, Mastering Genetics Flashcards
What is the function of restriction endonucleases in bacteria?
They provide a defense mechanism against infection by viruses.
Restriction endonucleases recognize and degrade viral DNA, thus preventing viral infections.
TRUE or FALSE?
Restriction endonucleases cut DNA at specific recognition sequences and then bond two strands covalently with the same “sticky ends.”
False
Restriction endonucleases cut DNA at specific sequences, but DNA ligase must be used to bond two strands covalently with the same “sticky ends.”
BamHI cuts the sequence 5′ G|GATCC 3′. Which of the following sequences would not be recognized by this enzyme?
BamHI cuts the sequence 5′ G|GATCC 3′. Which of the following sequences would not be recognized by this enzyme?
This sequence does not contain the BamHI recognition site.
Which of the following DNA sequences is one strand of a restriction enzyme recognition sequence?
5’ AAACCC 3’
5’ GGGTTT 3’
5’ GGGGGG 3’
5’ GGATCC 3’
5’ GGATCC 3’
The 5’ → 3’ sequence of the complementary strand would be the same as the 5’ → 3’ sequence of this strand; i.e., this sequence is symmetrical about the midpoint.
X-Gal is included in the growth medium on which cells transformed with bacterial plasmids are grown. The reason X-Gal is included is to _______.
identify bacteria that contain a recombinant plasmid
Colonies produced from cells containing a recombinant plasmid are white, whereas colonies from cells containing a nonrecombinant plasmid are blue.
TRUE or FALSE?
Within a six-base DNA recognition sequence, an enzyme that cuts between the 3rd and 4th bases from the 5’ end will generate blunt ends.
True
Restriction endonucleases are especially useful if they generate “sticky” ends. What makes an end sticky?
single-stranded complementary tails
Assume that a given plasmid vector to be used in a cloning experiment contains 4000 base pairs of DNA. Assume also that the restriction endonuclease Cuj cuts this plasmid at the following sites (starting from an arbitrary zero point): 1000, 1500, and 3000.
Given complete digestion of the plasmid with the endonuclease so that only linear fragments are produced, what sizes of DNA are expected?
500 bp, 1500 bp, and 2000 bp
TRUE or FALSE?
To isolate a bacterium with a plasmid that carries a desired DNA fragment cloned within the ampicillin resistance gene, we should grow bacteria in a medium that contains ampicillin.
False
TRUE or FALSE?
In general, the main goal of cloning is to include as many different genes as possible in a single cloning vector.
False
TRUE or FALSE?
A common term for a plasmid or other DNA element that serves as a cloning vehicle is vector.
True
Following are 4 processes common to most cloning experiments:
A) transforming bacteria
B) plating bacteria on selective medium
C) cutting DNA with restriction endonucleases
D) ligating DNA fragments
Place components of this list in the order in which they would most likely occur during a cloning experiment.
- Cutting DNA with restriction endonucleases.
- Ligating DNA fragments.
- Transforming bacteria.
- Plating bacteria on selective medium.
Some vectors such as pUC18 and others of the pUC series contain a large number of restriction enzyme sites clustered in one region. Which term is given to this advantageous arrangement of restriction sites?
multiple cloning site
What term is used to refer to the process in which DNA can be introduced into host bacterial cells?
transformation
Assume that one conducted a typical cloning experiment using a typical plasmid, transformed an appropriate host bacterial strain, and plated the bacteria on an appropriate X-gal medium. Blue and white colonies appeared. Which of the two types of colonies, blue or white, would most likely contain the recombinant plasmid?
white colonies
TRUE or FALSE?
Phage λ can carry larger DNA fragments than plasmids.
True
Phage vectors can carry DNA fragments of about 20 kb, whereas plasmids can only carry DNA of less than 15 kb.
Which of the following elements is not found in a plasmid?
Lambda arms
Lambda arms are regions that flank the inserted foreign DNA in phage λ vectors.
A DNA fragment is introduced into the lacZ gene of a plasmid, which also contains a tetracycline resistance gene. What is the appearance of bacteria transformed with this plasmid if they are spread on plates containing tetracycline and Xgal?
White colonies that are resistant to tetracycline
The presence of blue colonies means that the plasmid taken up by these bacteria is recombinant, since the lacZ gene was disrupted.
Why are filters overlaid with X‑ray film when screening a cDNA library?
To visualize probe hybridization events
The X‑ray film allows probe hybridization events on the filter to be visualized if a radioactive probe is used.
TRUE or FALSE?
Chromosome walking is used mainly to isolate a gene when the sequence of the gene is known.
False
Chromosome walking is used mainly when the gene of interest has not yet been cloned but its approximate location on a chromosome is known.
One of the primary reasons for generating a large number of clones in a eukaryotic genomic library is that ________.
each vector can take up only a relatively small fraction of the eukaryotic DNA
Which type of DNA library represents the genes expressed by a given cell at a certain time?
cDNA
cDNA libraries represent expressed genes from specific cell types under specific conditions. Noncoding sequences are not included.
Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot, one generally ________.
hybridizes filter-bound DNA with a DNA probe
Northern blots are used to study what type of molecule?
RNA
RNA fragments are separated using gel electrophoresis and blotted onto a nylon membrane. Labeled nucleic acid fragments are used to probe the filter and identify hybridizing sequences.