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: Hind III and Bgl I endonucleases will be used to cleave a plasmid into discrete fragments. The fragments will then be separated by agarose gel electrophoresis and analyzed. The relative locations (mapping) of the cleavage sites in the plasmid will then be determined.

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.

5 '-A |A G C T T-3' 3 '-T T C G A| A-5' Hind III (Haemophilus influenzae) 5- G C C N N N N / N G G C C G G N / N N N N C C G -5 BglI (Bacillus globigii)

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 counterclockwise direction.

The triple digest, A + B + C is a confirmatory test Generally, a restriction enzyme map is constructed by first 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).

Gel electrophoresis 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.

After electrophoresis, the DNA bands are visualized, and the migration distances of the known and unknown fragments are measured by Standard DNA fragments.

The standard fragments are used to make a standard curve on semi-log graph paper by plotting their sizes on the y-axis versus the migration distance on the x-axis.

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 insuffi cient 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.

Circular DNAs such as plasmids are supercoiled. Supercoiled forms of DNA have a more compact and entangled shape (like a twisted rubber band) than their corresponding non- supercoiled forms (linear, nicked and relaxed circles).

When supercoiled DNA is cleaved by a restriction enzyme once on each of the two strands, it unravels to its linear form. If supercoiled DNA is nicked (a phosphate bond is broken anywhere in the molecule, in either strand) it unravels to form a nicked circle (Figure 6).

supercoiled DNA migrates faster than its linear form migrates faster than its nicked circular form.

most preparations of uncut plasmid contain at least two topologically- different forms of DNA, corresponding to supercoiled forms and nicked circles.

Catenanes: During replication, several plasmid molecules can form interlocking rings with themselves. These forms are called catenanes. Catenanes can contain 2 plasmid molecules (dimer) Three molecules (trimer), etc. (Figure 7).

Catenanes migrate more slowly than single circular DNA that are nicked during electrophoresis. Dimers migrate faster than trimers, which migrate faster than tetramers, etc. Catenanes give rise to the same final restriction enzyme cleavage patterns as their uncatenated (single circular) forms.

The The Experiment In this experiment you will determine the locations of restriction enzyme cleavage sites on a circular plasmid DNA. Standard DNA fragment sizes are provided to construct the standard curve. The enzymes used to cleave the plasmid are Hind III and Bgl I. Assume the Bgl I site is at position 0. The objective is to calculate the distances between the points of cleavage and to determine their relative orientations.

GENERAL INSTRUCTIONS Add enzyme(s), reaction mixtures to the DNA to start the reactions. Use a fresh micropipet tip to dilute the DNA or enzymes. 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.

PREPARING THE GEL FOR ELECTROPHORESIS

Size Determination of DNA Restriction Fragments

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