B

BamHI

Pst I

Pst I

PstI

■ Figure 6-2 Two possible maps inferred from the observations described in Figure 6-1. The BamHI site positions fragment A at one end (or the other) of the map. Determination of the correct map requires information from additional enzyme cuts.

BamHI

Pst I

Pst I

PstI

■ Figure 6-2 Two possible maps inferred from the observations described in Figure 6-1. The BamHI site positions fragment A at one end (or the other) of the map. Determination of the correct map requires information from additional enzyme cuts.

the order of the restriction fragments, another enzyme is used, for example BamHI. Cutting the same fragment with BamHI yields two pieces, indicating one BamHI site in this linear fragment (see Fig. 6-1). Observe that one restriction product (F) is very much larger than the other (E). This means that the BamHI site is close to one end of the fragment. When the fragment is cut simultaneously with PstI and BamHI, five products are produced, with PstI product A cut into two pieces by BamHI. This indicates that A is on one end of the DNA fragment. By measuring the number and length of products produced by other enzymes, the restriction sites can be placed in linear order along the DNA sequence. Figure 6-2 shows two possible maps based on the results of cutting the fragment with PstI and BamHI. With adequate enzymes and enzyme combinations, a detailed map of this fragment can be generated.

Mapping of a circular plasmid is slightly different, as there are no free ends (Fig. 6-3). The example shown in the figure is a 4-kb pair circular plasmid with one BamHI site and two XhoI sites. Cutting the plasmid with BamHI will yield one fragment. The size of the fragment is the size of the plasmid. Two fragments released by XhoI indicate that there are two XhoI sites in the plasmid and that these sites are 1.2 and 2.8 kb pairs away from each other. As with linear mapping, cutting the plasmid with XhoI and BamHI at the same time will start to order the sites with respect to one another on the plasmid. One possible arrangement is shown in Figure 6-3. As more enzymes are used, the map becomes more detailed.

The pattern of fragments produced by restriction enzyme digestion can be used to identify that DNA and to monitor certain changes in the size, structure, or sequence of the DNA. Because of inherited or somatic dif

BamHI XhoI

BamHI +

XhoI

BamHI XhoI

BamHI +

XhoI

■ Figure 6-3 Restriction mapping of a plasmid. After incubating plasmid DNA with restriction enzymes, agarose gel electrophoresis banding patterns indicate the number of restriction sites and the distance between them.

BamHI

XhoI

BamHI

XhoI

XhoI

■ Figure 6-3 Restriction mapping of a plasmid. After incubating plasmid DNA with restriction enzymes, agarose gel electrophoresis banding patterns indicate the number of restriction sites and the distance between them.

ferences in the nucleotide sequences in human DNA, the number or location of restriction sites for a given restriction enzyme are not all the same in all individuals. The location and order of restriction enzyme sites on a DNA fragment is a molecular characteristic of that DNA. The resulting differences in the size or number of restriction fragments are called restriction fragment length polymorphisms (RFLPs). RFLPs were the basis of the first molecular-based human identification and mapping methods. RFLPs can also be used for the clinical analysis of structural changes in chromosomes associated with disease (translocations, deletions, insertions, etc.).

Hybridization Technologies

Procedures performed in the clinical molecular laboratory are aimed at specific targets in genomic DNA. This requires visualization or detection of a specific gene or region of DNA in the background of all other genes. There are several ways to find a particular region of DNA from within an isolated DNA sample. The initial method for molecular analysis of specific DNA sites within a complex background was the Southern blot. Modifications of the Southern blot are applied to analysis of RNA and protein in order to study gene expression and regulation (Table 6.1).

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