Experiment
2.2 Restriction Endonuclease Digestion
(version 01/31/00
)
Background
Restriction endonucleases are enzymes that cut DNA at specific sites based on nucleotide sequence. Useful sources of information on restriction enzymes can be found on the web sites of commercial producers such as New England Biolabs.
The purpose of this laboratory exercise is to use restriction enzymes to confirm the sequence of the plasmid (pYP) purified in the previous laboratory. Students will cut their purified plasmid with a series of restriction enzymes, alone and in combination. The lengths of the products of the digestion reactions will be used to construct a "restriction map". A restriction map gives relative positions of cutting sites. The restriction map will confirm the identity and sequence of the plasmid. Some of the restriction sites are within the tRNA (or tRNA mutant) coding sequence.
The company that supplied the pDEFEND2 vector (New England Biolabs) provided information on the sequence of the polylinker region and on the position of the T7 RNA polymerase promoter. They reported that pUC19 is the parent construct (i.e., that pDEFEND2 was made from pUC19). NEB did not provide the complete sequence of the pDEFEND2 vector.
Prelab
1) Write a brief synopsis of the procedure in point form.
2) For the restriction enzymes EcoR I, BssH II, Dra I, Ear I, and Hae II, use the information provided by NEB to determine the recognition sequences and positions of cutting sites relative to the recognition sequences.
3) Use webcutter to find all the restriction sites for EcoR I, BssH II, and Hae II in pUC19. Copy the pUC19 sequence into the webcutter window marked "Paste the DNA sequence into the box below." Select the appropriate restriction enzymes. Include a condensed version of the printout with your report. A part of the web cutter output for analysis of restriction enzyme Dra I follows. The output indicates that Dra I cuts pUC19 3 times, at positions 1565, 1584, and 2276, that the recognition sequence is 5'TTTAAA3', and that the cut site is on the 5' side of the the T.
Webcutter Output
Table by Enzyme Name
Enzyme No. Positions Recognition
name cuts of sites sequence
Dra I 3 1565 1584 2276 ttt/aaa More info<
4) Make a table of anticipated fragment sizes if pUC19 is cut with (a) EcoR I, (b) Dra I, (c) Hae II, (d) EcoR I plus Dra I, (e) EcoR I plus Hae II, and (f) Dra I plus Hae II. Include a condensed version of the printout with your report. .
5) Use a word processing program to construct a hypothetical sequence for pYP. Note that we do not know the complete sequence. But we do know that the parent vector is pUC19. And we know the sequences of the T7 RNA polymerase promoter, and of the inserted coding region.
6) Use webcutter to predict all the restriction sites for EcoR I, BssH II, and Hae II in your proposed sequence of pYP-GCAA. Make a table of anticipated fragment sizes if pYP-GCAA is cut with (a) EcoR I, (b) BssH II, (c) Hae II, (d) EcoR I plus BssH II, (e) EcoR I plus Hae II, and (f) BssH II plus Hae II
Unlinearized plasmid pYP
Ear I Linearized plasmid pYP
BSA (bovine serum albumin)
Eco RI Restriction Enzyme
10X NEBuffer EcoR I (10x: 50 mM
NaCl, 100 mM Tris-HCl, 10 mM MgCl2, 0.025% Triton X-100 (pH 7.5 @ 25°C))
Hae II Restriction Enzyme
10X NEBuffer 4 + BSA (10x: 50 mM
NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol (pH 7.9 @ 25°C,
Supplement with 100 microgram/ml BSA.)
BssH II Restriction Enzyme
10X NEBuffer BssH II (10x: 100 mM
NaCl, 10 mM Bis Tris Propane-HCl, 10 mM MgCl2, 1 mM dithiothreitol (pH
7.0 @ 25°C))
Samples from preceding plasmid prep experiment.
DNA Size Markers
Ear I digest
The Ear I digest of pYP-GCAA was performed in the preceding laboratory exercise (the plasmid prep). Obtain the Ear I restriction digest from the TA. Use 10 microliters of the Ear I digest to run a gel for this experiment. The remainder will be used in the transcription reaction in the next laboratory exercise.
Eco RI Digest
To Sample E, add 1.5 microliters of the 10X NEBuffer EcoR I, 1 microliters of EcoR I enzyme, and 2.5 microliters dd H2O.
Hae II Digest
To Sample H, add 1.5 microliters of the 10X NEBuffer 4, 1 microliters of Hae II enzyme, and 2.5 microliters dd H2O.
BssH II Digest
To Sample B, add 1.5 microliters of the 10X NEBuffer BssH II, 1 microliters of BssH II enzyme, and 2.5 microliters dd H2O.
Digestion
Incubate each sample at 37 deg C for one hour.
The Agarose Gel
The procedure for setting up an agarose gel is given here.
Run 7 lanes on the gel
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Results and Discussion
Address the following.
1) Construct a calibration curve for your agarose gel by graphing migration distance vs. log (number of base pairs) for the molecular weight standards. The TA will indicate the sizes of the marker DNA fragments.
2) Determine the number of fragments and the approximate size of each fragment (in base pairs) resulting from each restriction enzyme digest.
3) Assign as many of the bands on the gel as possible.
4) Describe differences between nicked, cut and supercoiled plasmids.
5a) Assuming pYP is in the B-conformation, what is Lko (the relaxed linking number)?
5b) If the superhelical density (s) of pYP is -0.09, then what is Lk (the linking number?
5c) If the superhelical density of pYP is -0.09, and tw (the twist) is 324, then what is wr (the writhe)?
5d) What is a topoisomer?
6) What is the roll of ethidium in this experiment?
7) How does ethidium bind to DNA? Does ethidium form hydrogen bonds with DNA?
8) Define "sticky ends".
9a) Give two examples of sequence-specific hydrogen bonding interactions between Eco RI and DNA in the precleavage complex. To specify each interaction give pairs of atom names (one from the DNA, one from the protein). Give the residues names and residue numbers. Specify which atom is the hydrogen bond acceptor, and which is the donor. Give the distance between the atoms that are interacting. In this context "sequence-specific" indicates that if one changes the DNA sequence, the nature of the interaction will change. So a single hydrogen bond from a lysine to the N7 of adenine or to a phosphate oxygen is not sequence-specific. Switching an adenine to a guanine will not change either of those hydrogen bonding interactions.
9b) For each sequence-specific interaction (above) use RASMOL to export a figure that clearly illustrates the interaction. Include the figure in your discussion.
10) Using the webcutter and the pYP sequence, suggest additional restriction enzymes that could be used to help confirm that the mutant tRNA coding sequence is contained within pYP. Describe the anticipated results of digestion with these restriction enzymes.
11) How many recognition sites for a restriction enzyme with a hexanucleotide recognition sequence would you anticipate in a random-sequence DNA fragment the size of pDEFEND2 (3191 BP)? How many in a DNA fragment the size of the E. coli genome (about 4 x 106 BP)? Explain. (Hint: See Voet & Voet, pp. 825-830).
