EXPERIMENT OBJECTIVE:. The objective of this experiment is to develop an understanding of DNA mapping by determining restriction enzyme cleavage sites on a circular DNA plasmid. BRIEF DESCRIPTION OF EXPERIMENT:.
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Restriction enzymes are endonucleases that cleave both strands of DNA at specifi c sequences of bases. The cleavage site is usually located within or near the recognition site. The location of restriction enzyme
cleavage sites are important in analysis, mapping of genetic structure and in molecular cloning experiments.
Restriction enzyme mapping determines the relative positions of cleavage sites to one another in a DNA molecule. This is done by determining sizes of DNA fragments generated by different combinations of restriction enzyme digests.
As an example, consider a 5000 base pair, circular plasmid DNA containing single recognition sites for enzymes A, B, and C. Any one of these enzymes will cleave the DNA once to produce a linear molecule of 5000 base pairs.
Differently paired combinations of enzymes in the same reaction mixture (double-digests) will produce the following DNA fragments (sizes in base pairs):
Arbitrarily placing one of the cleavage sites at the top of a circle. This site acts as a reference point.
The closest cleavage site to this point can be placed in a clockwise or
The triple digest, A + B + C is
a confirmatory test
Generally, a restriction enzyme map is constructed by fi rst determining the number of fragments each individual enzyme produces. The size and number of fragments is determined by electrophoresis.
Determination of site order requires choosing one of the cleavage sites as an arbitrary starting point at 12 o'clock on a circle (position 1).
Usually, this is an enzyme that has a single cleavage site in the DNA.
Using the shortest distances between sites, as determined in the double digests, the sites are placed on a circle (or a line,
depending on the DNA).
The pores in the gel separate the DNA molecules according to their size and shape. The migration rate of DNA molecules having the same shape is inversely proportional to their size. This means that the smaller the DNA molecule, the faster it migrates through the gel.
For every base pair (average molecular weight of approximately 660) there are two charged phosphate groups. Therefore, the net charge is accompanied by approximately the same mass.
The absolute amount of charge on the molecule is not a critical factor in the separation
process. The separation occurs because smaller molecules pass through the pores of the gel more easily than larger ones, i.e., the gel primarily separates linear pieces of DNA based on physical size.
If a DNA molecule contains several recognition sites for a restriction enzyme, then under certain experimental conditions, it is possible that certain sites are cleaved but not others. These incompletely cleaved fragments of DNA are called partial digests
(partials). Partials can arise if an insufficient amount of enzyme is used or the reaction is stopped after a short time (Figure 5). Reactions containing partials may also contain some molecules that have been completely cleaved.
Most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles.
• Mix by tapping the tube after transfer of reagents.
• Incubate reactions at 37°C in a waterbath. Reaction tubes can be suspended in a test tube rack that is partially immersed.
• Gel loading solution contains protein denaturants that stop the enzyme reactions. Do not place a pipet that has been in gel loading solution into a tube containing enzymes until you are ready to terminate the reaction.