Restriction Mapping of Plasmid DNA
Introduction
The primary tools used by the molecular geneticist in manipulating DNA are restriction enzymes (1st discovered in the 1960's by Dr. Arber). Because a restriction enzyme reaction is very specific for a given DNA sequence, whole genomes can be divided into smaller, discrete pieces of DNA and these enzymes are extensively used in recombinant DNA technology.
Over the years, the number and uses of these enzymes have increased as new molecules have been discovered, our understanding of the details of the enzymatic reactions has improved, and many commercial manufacturers of products supporting molecular genetics have emerged to guarantee the widespread availability of these reagents. With these advances, the analysis of genes has been both simplified and expanded in scope.
Restriction enzymes bind specifically to cleave double-stranded DNA at specific sites within or adjacent to a particular sequence known as the recognition sequence or restriction site. The vast majority of restriction enzymes recognize specific sequences that are four, five or six nucleotides in length and display twofold symmetry. A few enzymes, however, recognize longer sequences that are degenerate. The location of cleavage sites within the axis of dyad symmetry differs from enzyme to enzyme. Some restriction enzymes cleave both strands exactly at the axis of symmetry, generating fragments of DNA that carry blunt ends; others cleave each strand at similar locations on opposite sides of the axis of symmetry, creating fragments of DNA that carry protruding single-stranded termini.
If the restriction enzyme cleaves each strand of the substrate DNA on the 5' side of the axis of dyad symmetry, the resulting staggered break yields fragments of DNA that carry protruding cohesive 5' termini. For example, in today's lab we will cut your isolated plasmid with EcoRI that recognizes the sequence:
GAATTC
CTTAAG
in double-stranded DNA and cleaves it as follows:
5'NNNNNG//AATTCNNNNN3'
3'NNNNNCTTAA//GNNNNN5'
The hydrogen bonding of the four bases between the sites of cleavage is not favored under these conditions used for digestion. Therefore, the original segment of DNA separates into two fragments:
5'NNNNNG______pAATTCNNNNN3'
3'NNNNNCTTAAp______GNNNNN5'
If fragments bearing compatible protruding termini are incubated under conditions that favor formation of base pairs, they can anneal with one another.Since all DNA fragments created by cleavage with EcoRI carry the same protruding 5' termini, these fragments can be joined in novel combinations (i.e. recombinant DNA technology). In fact, EcoRI was the first enzyme used to create recombinant DNA molecules in the 1970's.
DAY ONE - Setting up a restriction digest
Different manufacturers of restriction enzymes recommend significantly different digestion conditions, even for the same restriction enzyme. Because most manufacturers have optimized the reaction conditions for their particular preparations, it is recommended to follow the instructions on the information sheets supplied with the enzymes. Most manufacturers also supply concentrated buffers (usually 10X stock solutions) that have been tested for efficacy with each batch of purified enzyme. Buffers for different restriction enzymes differ chiefly in the concentration of NaCl that they contain. These buffers should be used whenever possible.
In today's lab, you will first set up an EcoRI restriction digest of your plasmid DNA. In general, all restrictions digests contain four components: DNA (e.g. plasmid DNA), restriction enzyme buffer, water, and restriction enzyme. It is very important to add the components in the following order: water, DNA, buffer, and then enzyme. It is also very important to use separate, sterile pipet tips for each transfer!
Remember; restriction enzymes are very expensive and should be used in a careful manner!
Although the volume of a restriction enzyme reaction is not constant, the total volume of most reactions is between 10 and 50 microliters. In today's exercise, you will set up a 24 microliter restriction enzyme reaction (digest).
First, you must determine the amount of plasmid DNA in your sample (from last week's exercise - ask your instructor for assistance!). A typical reaction contains 0.2 - 1 micrograms of DNA.
1. Place the DNA solution in a sterile microfuge tube and mix with sufficient sterile water to give a volume of 17 microliters.
2. Add 2 microliters of 10X EcoRI restriction digestion buffer. Mix by lightly tapping the tube.
3. Add 1 microliter (10 - 20 units) of EcoRI restriction enzyme. Mix by lightly tapping the tube.
Note: Make sure that you pipet each liquid into the bottom of the tube. Change pipet tips before each transfer.
One unit of enzyme is usually defined as the amount required to digest 1 microgram of DNA to completion in 1 hour in the recommended buffer and at the recommended temperature in a 20 microliter reaction. In general, digestion for longer periods of time or with excess enzyme does not cause problems unless there is contamination with nucleases. Such contamination is rare in commercial preparations of restriction enzymes.
4. Incubate the microfuge tube for 2 hours at 370C.
5. Your instructor will stop the reaction by adding 1 microliter of 0.5M EDTA (pH 8.0) to the microfuge tube. Your instructor will store the microfuge tube at 40C until the next laboratory period.
DAY TWO - Restriction mapping of plasmid DNA
Generally, the first step in exploring a cloned DNA fragment is to construct its restriction map. By digesting the DNA with various restriction enzymes, alone and in combination, one can determine the number and relative positions of cutting sites along the DNA for each restriction enzyme. pBluescribe (Stratagene) is 2746 base-pairs in length and contains one EcoRI restriction site. Your plasmid, pO.380, contains approximately 3800 base pairs of Drosophila DNA that was inserted into the EcoRI site within pBluescribe. Thus, an EcoRI restriction digest of pO.380 should cut this circular recombinant plasmid into two fragments. One fragment will be approximately 2.7 kilobases (kb) in length (pBluescribe) and the second, and larger, fragment should be 3.8 kb in length (Drosophila DNA).
Remember from last week's lab that an important application of gel electrophoresis is the determination of molecular size. Molecules of linear double-stranded DNA migrate through agarose gels at rates that are inversely proportional to the number of base pairs. Thus, the 2.7 kb EcoRI restriction fragment should migrate faster through an agarose electrophoretic gel relative to the 3.8 kb DNA fragment.
In today's lab, you will set up a 0.8% agarose gel (follow the procedure that was used in last week's exercise). You will run your two samples on this gel. One sample will be your EcoRI-digested plasmid DNA. Your second sample contains a series of DNA fragments of known length (your standard is bacteriophage lambda DNA (linear virus approximately 48,502 base pairs digested with the restriction enzyme HindIII). This digestion produces a series of DNA fragments with the following lengths (in bp): 23,130; 9,416; 6,557; 4,361; 2,322; 2,027, 564, and 125.
Linear DNA fragments (fragments resulting from restriction digests) migrate at rates inversely proportional to the log10 of their molecular weight. For simplicity's sake, base-pair length is substituted for molecular weight.
Photograph your stained gel under UV light. Use the photograph and a ruler to construct a graph that relates the base-pair size of each HindIII lambda restriction fragment to the distance migrated through the gel.
Set up a semilog graph using the distance migrated as the X (arithmetric) axis and the base-pair length as the Y (logarithmic) axis.
Now calculate the size of your EcoRI plasmid DNA fragments. Do they agree with the predicted sizes?
Sample Restriction Mapping Problem:
Digestion of a 4 kb DNA molecule with EcoRI yields two fragments of 1 kb and 3 kb each. Digestion of the same molecule with HindIII yields fragments of 1.5 kb and 2.5 kb. Finally, digestion with EcoRI and HindIII in combination yields fragments of 0.5 kb, 1 kb, and 2.5 kb. Draw a restriction map indicating the positions of the EcoRI and HindIII cleavage sites.