Quiz 9: Restriction Enzymes
The recognition site for EcoRI is GAATTC. What would be the average size of the restriction fragments generated in a restriction digest of human genomic DNA?
4096 bp
Some restriction endonucleases are referred to as four-cutters. What makes an enzyme a four-cutter and how frequently would you expect a four-cutter to cut DNA?
Four-cutters recognize specific nucleotide sites that are four nucleotides long and cut about once every 256 bp on average.
If you discovered a bacterial cell that contained no restriction endonucleases, which of the following would you expect to happen? The cell would be easily killed by bacteriophages. The cell would become an obligate parasite. The cell would create incomplete plasmids. The cell would be unable to replicate its DN
a
Which of the following is NOT important when planning your RE digest? The sequence of DNA upstream of your MET gene in the yeast genome. The sequence of the lacZ gene. The sequence of the pBG1805 plasmid, including it's URA3 gene and origins of replication. The sequence of your MET gene
a
An investigator digests bacteriophage DNA with three different restriction endonucleases in separate reactions. One reaction contains Pst I, which recognizes the sequence TTATAA. The second reaction contains HaeIII, which recognizes the sequence GGCC, and the third reaction contains Not I, which recognizes the sequence GCGGCCGC. The products of the three reactions are separated on an agarose gel. Which of the reactions would you expect to generate the smallest number of fragments? They would all be expected to produce the same number of fragments. Not I Pst I Hae III
b
Many restriction endonucleases recognize palindromes in DNA. Which of the following DNA sequences is a palindrome? (Remember that DNA is a double helix with two antiparallel strands. Only one strand of the helix is shown.) GATTAG GATATC GATGAT GATCGA
b
Which of the following is true about restriction endonucleases (REs)? A hypothetical RE that is a 3 cutter would cut every ~32 base pairs in a sequence. Bacterial cells can contain multiple REs that recognize different DNA sequences. Bacterial species are named for the REs that are present in their cytoplasm. Most commercially available REs are purified from their species of origin.
b
Restriction endonucleases (REs) are often used to fingerprint DNA. Which of following statements are true? Restriction endonucleases nick one strand of the DNA helix, generating nicked fragments that can be separated by electrophoresis. Cleavage sites for a particular RE are regularly spaced along a DNA molecule, separated by an interval characteristic of the particular RE.! The lengths of the DNA fragments produced by a particular RE vary widely. Restriction endonucleases are robust enzymes that resist denaturation under conditions that denature DNA.
c
Like all enzymes, restriction endonucleases (REs) need to be handled very carefully to ensure that they do not denature. Which of the following is NOT recommended to protect these fragile structures from denaturation? REs should be stored in glycerol solutions, which do not freeze at temperatures below the freezing point of water. Investigators should minimize the number of air bubbles introduced into RE solutions during pipetting. REs should be stored at low temperatures, since the rate of denaturation increases with temperature. REs activity is best preserved by freezing the RE and thawing it out for brief periods of time as needed.
d
Bacteriophage lambda has a 48 kb linear genome. If you digest lambda DNA with an enzyme known to recognize three sites in its sequence, how many bands would you expect to see on an agarose gel? (Our MW standards are derived from restriction digests of lambda DNA.)
four