biology : class12 Restriction Enzyme Methylation : DNA Isolation, Restriction Enzymes, Vector, Ligation, Host Cell : Recombinant DNA Technology MCQ Questions and Answers
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Restriction Enzyme Methylation
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MCQ Questions
1. A plasmid contains an unmethylated recognition site for a restriction enzyme, but the same site is methylated by a host DNA methyltransferase. If the enzyme is methylation-sensitive, what is the most likely outcome?
A.
The enzyme cleaves the site more rapidly than usual
B.
The enzyme fails to cleave or cleaves very inefficiently at the site
C.
The methyl group is removed by the restriction enzyme before cleavage
D.
The enzyme cleaves only the opposite strand at the site
Correct Answer:
The enzyme fails to cleave or cleaves very inefficiently at the site
Explanation:
Methylation of bases within or near a restriction-recognition sequence can prevent a methylation-sensitive restriction enzyme from binding productively or cutting the DNA.
2. Why do restriction-modification systems commonly protect bacterial genomic DNA from cleavage by their own restriction enzymes?
A.
The host methyltransferase methylates the restriction sites in the bacterial genome
B.
The host DNA polymerase removes all restriction sites during replication
C.
The restriction enzyme is synthesized only after foreign DNA is degraded
D.
The bacterial genome contains no sequences recognized by restriction enzymes
Correct Answer:
The host methyltransferase methylates the restriction sites in the bacterial genome
Explanation:
In a restriction-modification system, a cognate methyltransferase modifies the host's recognition sequences, allowing the restriction enzyme to distinguish protected self-DNA from unmethylated foreign DNA.
3. A plasmid propagated in an Escherichia coli strain deficient in Dam methyltransferase is digested with a Dam-sensitive restriction enzyme. Compared with the same plasmid propagated in a Dam-positive strain, what is expected?
A.
The plasmid from the Dam-deficient strain is more resistant to digestion
B.
The plasmid from the Dam-deficient strain is more readily cleaved at GATC sites
C.
Both plasmids are necessarily identical in their methylation at GATC sites
D.
Only the RNA transcript of the plasmid is affected by Dam deficiency
Correct Answer:
The plasmid from the Dam-deficient strain is more readily cleaved at GATC sites
Explanation:
Dam methyltransferase methylates the adenine in GATC sequences. In its absence, these sites remain unmethylated and can be cleaved by enzymes whose activity is blocked by Dam methylation.
4. A restriction enzyme recognizes the sequence 5'-GATC-3' and cannot cut when the adenine is methylated. Which plasmid preparation is most suitable when complete digestion at these sites is required?
A.
Plasmid isolated from a host with active Dam methyltransferase
B.
Plasmid isolated from a host lacking Dam methyltransferase activity
C.
Plasmid isolated from a host with increased DNA ligase activity
D.
Plasmid isolated from a host with increased restriction-enzyme expression
Correct Answer:
Plasmid isolated from a host lacking Dam methyltransferase activity
Explanation:
A Dam-deficient host produces plasmid DNA with unmethylated GATC sites, allowing a Dam-sensitive enzyme to recognize and cleave those sites efficiently.
5. Two restriction enzymes recognize the same nucleotide sequence, but only one is inhibited by methylation at a particular base in that sequence. These enzymes are best described as:
A.
Isoschizomers with identical methylation sensitivity
B.
Isoschizomers that differ in methylation sensitivity
C.
DNA methyltransferases with identical cleavage specificity
D.
DNA ligases that differ in their ability to seal methylated DNA
Correct Answer:
Isoschizomers that differ in methylation sensitivity
Explanation:
Enzymes recognizing the same sequence are isoschizomers, but they may differ in whether methylation blocks cleavage. This difference is useful when selecting an enzyme for methylated templates.
6. A DNA fragment is methylated at a base outside a restriction enzyme's recognition sequence, and the enzyme is known to be unaffected by external methylation. What is the most reasonable prediction?
A.
Cleavage will be blocked because any methyl group prevents DNA cleavage
B.
Cleavage will occur normally because the relevant recognition sequence is not methylation-sensitive
C.
The methylated base will be converted into a restriction site
D.
The enzyme will cleave only after the methyl group is transferred to the opposite strand
Correct Answer:
Cleavage will occur normally because the relevant recognition sequence is not methylation-sensitive
Explanation:
Methylation affects restriction digestion only when its position and the enzyme's sensitivity are relevant. Methylation outside the recognition region generally does not inhibit an enzyme known to tolerate it.
7. A recombinant plasmid is cut with a restriction enzyme in a host strain but not when the same plasmid is prepared from another strain. Which explanation most directly accounts for this difference?
A.
The plasmid sequence necessarily mutated during purification
B.
The two host strains produced different methylation patterns at the enzyme's recognition sites
C.
The restriction enzyme changes its recognition sequence after each reaction
D.
The plasmid becomes single-stranded only in the second host
Correct Answer:
The two host strains produced different methylation patterns at the enzyme's recognition sites
Explanation:
Different host methyltransferases can modify the same plasmid differently. If the enzyme is sensitive to one of these modifications, digestion can differ even though the underlying plasmid sequence is unchanged.
8. A restriction enzyme is blocked by methylation of a recognition-site adenine on one strand. Why can methylation of the corresponding adenine on the complementary strand also influence digestion after DNA replication?
A.
Replication can convert a hemimethylated site into a fully methylated site recognized as protected
B.
Replication removes the recognition sequence from both strands
C.
Replication changes adenine into cytosine at every restriction site
D.
Replication permanently denatures the restriction enzyme
Correct Answer:
Replication can convert a hemimethylated site into a fully methylated site recognized as protected
Explanation:
After replication, methylation patterns can be maintained on newly synthesized DNA. A site that becomes methylated on both strands may be protected from a methylation-sensitive restriction enzyme.
9. A cloning vector is propagated in a host that methylates a restriction site needed for later insertion of an insert. What is the main practical consequence?
A.
The vector may resist digestion at the intended cloning site
B.
The vector will automatically ligate to any DNA fragment
C.
The methyl group will create an additional selectable marker
D.
The vector will lose all antibiotic-resistance genes during replication
Correct Answer:
The vector may resist digestion at the intended cloning site
Explanation:
If host-directed methylation occurs at a cloning site's recognition sequence and blocks the chosen restriction enzyme, the vector cannot be opened efficiently for ligation.
10. A researcher needs to distinguish two DNA samples containing the same restriction-recognition sequence but different methylation states. Which approach is most informative?
A.
Digest both samples with a methylation-sensitive enzyme and compare the cleavage patterns
B.
Incubate both samples only with DNA ligase and compare ATP consumption
C.
Measure the absorbance of both samples without enzymatic treatment
D.
Digest both samples with an enzyme known to recognize no DNA sequence
Correct Answer:
Digest both samples with a methylation-sensitive enzyme and compare the cleavage patterns
Explanation:
A methylation-sensitive restriction enzyme can cleave the unmethylated sample while failing to cut the methylated sample, producing distinguishable fragment patterns.
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