Understanding Plasmid DNA

    1. Know the main conformations of uncut plasmid DNA and be able to recognize them on a gel.

    2. Be able to recognize the major differences between bulk prep and miniprep plasmid DNA.

Conformations of Uncut Plasmid DNA

    We analyze DNA that has been cut with restriction enzymes by determining fragment sizes using agarose gel electrophoresis. However, uncut plasmid DNA has several distinct conformations which can be identified when the uncut plasmid is electrophoresed in an agarose gel.

Supercoiled DNA (Form I), is the fastest conformation of the uncut plasmid. The enzyme DNA gyrase introduces these extra twists into chromosomal and plasmid DNA of bacteria. The bacteria use the superhelical tension to assist in processes like replication and transcription. After isolating the supercoiled (sc) plasmid DNA, it is wound up into a compact structure. Imagine taking a circle of string and rolling it around in your hands until it forms a little ball. Because of its compact shape, sc DNA is the fastest moving comformation in the gel.

Nicked Circle DNA (Form II) is also called relaxed circle. In bacteria, the enzyme topoisomerase I will nick one strand of the helix so that DNA polymerase has access to the DNA for replication. Once one of the strands has been cut, the superhelical tension relaxes and the tightly-wound ball becomes a floppy circle. A nick can also occur during isolation of the plasmid because of enzyme activity or mechanical shearing of the DNA.  Nicked circle (nc) is the slowest conformation of uncut DNA.

Linear DNA (Form III) is produced when a restriction enzyme cuts a plasmid at only one site. Both strands of the helix are cut at the same place. Linear DNA can occur because of endonuclease contamination of the isolated plasmid, or because of harsh treatment. On a gel the linear DNA will run between the sc and nc conformations, often closer to the sc band.
 

The Figure to the right shows very overloaded lanes of uncut pAMP and pKAN DNA (how do we know?). However, because there is so much DNA, all the conformations are visible.

The Figure to the right gives another example of the conformations of uncut plasmid DNA. Which plasmid is larger, the pAMP or the pKAN? How can you tell?

Small-scale and Large-scale Preparation of Plasmid DNA

    Depending on the need, plasmid DNA can be isolated in different quantities. Small-scale preparation of plasmid DNA is often termed a "miniprep" and starts with 1-5 ml of an overnight bacterial culture. There are many different miniprep protocols available. Some protocols are variations on the original protocol published by Birnboim and Doly (Nucleic Acids Research7; 1513, 1979). Others use recent technology and employ a resin which binds, and then elutes, purified plasmid DNA. Several micrograms of DNA can be isolated with the miniprep, and the procedure can be completed in a relatively short time. Minipreps are often used to detect successful recombinants after the potential recombinant DNA has been inserted into bacteria (the process of transformation).

    Many micrograms of DNA can be isolated using a large-scale or "bulk prep" protocol. These protocols use from 25 to several hundered ml of bacterial culture. Formerly, large-scale isolation of plasmid DNA required many hours of ultacentrifugation in a solution of cesium chloride-ethidium bromide. This procedure was time-consuming and required handling of toxic materials. Now, the same resins that can be used for the miniprep are commonly employed for bulk preparation of the plasmid DNA and the procedure can be completed in several hours.
 
 

Differences Between Bulk Prep and Miniprep DNA

    While all the bulk prep procedures result in high-quality plasmid DNA if performed properly, there are often differences in the quality of miniprep DNA. The miniprep may contain pieces of sheared chromosomal DNA and various proteins. Minipreps usually contain RNA, so RNAse is added at some pont during purification. (When is RNAse added in our protocol?  Which samples receive the RNAse?). Finally, there may be salts or traces of the miniprep reagents in the isolated DNA.

    Because of these differences in purity, the miniprep DNA looks and cuts differently than the bulk prep plasmid. Miniprep DNA can be more difficult to cut because associated proteins may interfere with recognition of the cleavage site, and contaminants can result in sub-optimal reaction conditions in the restriction digest. Thus, it is more common to see a partial or incomplete digest with miniprep DNA. The RNA is able to bind ethidium bromide. If it is not degraded by addition of RNAse, it will form a diffuse "RNA cloud" towards the bottom of the gel. If you are trying to detect a small fragment from a restriction digest, it can be obscured by the RNA cloud. Finally, the gel lanes of miniprep DNA are often dark or smeared. This contrasts with the clear wells and gel lanes of bulk prep DNA, except for the presence of discrete DNA bands.
 
 
 

The Figure at the right shows several samples of miniprep DNA. M1 and M2 stand for miniprep one and miniprep two, respectively. The (+) or (-) represent WITH or WITHOUT restriction enzymes. Note the dark wells and the smear in some of the lanes. In this case, all the minipreps show complete digests.