0.2 Eukaryotic genetics  (Page 7/9)

In the human genome, the sites that have the properties most favorable to such extensive variation include a repetition of the same short DNA sequence a variable number of times. Such sequences are called tandem-repeat sequences. A DNA sequence with such variation may be as short as two base pairs or as long as several hundred base pairs. Highly variable sequences of this type are well distributed throughout the length of every human chromosome. When tandemly repeated sequences are replicated during cell division, the number of repeats can change. The frequency of this kind of replication error is high enough to make alternative lengths at the polymorphic site common, but the rate of change in the length of the site is low enough that the size of the DNA at the polymorphic site serves as a stable trait in family studies ( Figure 1A ).

Two techniques, Southern blotting and the polymerase chain reaction (PCR), can measure the length of the DNA sequence at the polymorphic site ( Figure 1B ). The one to choose depends on the length of the tandemly repeated sequence. A repeated sequence 20 to 40 base pairs in length leads to variation in DNA lengths of hundreds or even thousands of base pairs at the polymorphic site. Southern blotting is best for visualizing this degree of variation in length. Very short tandemly repeated sequences, only two, three, or four base pairs long, can also vary highly. For these, the PCR is preferred. Whichever technique is used, its goal is to assess accurately the length of the DNA segment between two fixed points on each chromosome. These two points include some DNA adjacent to the repeated sequence as well as the repeated sequence itself. In the case of Southern blotting, the position of the fixed points is defined by the location of restriction-enzyme digestion sites in the DNA flanking the repeated sequence. In the case of PCR, the positions in the flanking DNA of sequences homologous to the oligonucleotide PCR primers define the fixed points.

In Southern blotting, the DNA isolated from each patient or tumor to be typed is digested with a restriction enzyme, separated on the basis of size by agarose-gel electrophoresis, and transferred to a nylon membrane. A DNA probe can reveal directly on the nylon membrane the size of DNA fragments carrying the repeated sequence. This probe corresponds to a sequence in the DNA flanking the repeated sequence. In general, DNA from one person shows two such DNA fragments or bands ( Figure 1C ). For each chromosomal site, one of the two bands will be passed to the next generation, and the other will not, thus indicating the outcome in genetic transmission that occurred at this particular chromosomal site.

With the PCR method, the unique sites of primer binding adjacent to the repeated sequence allow specific amplification of the region that includes the repeat. The size of the amplified DNA molecules representing the polymorphic site can now be determined with the same technique that determines the DNA sequence. Precise determination of the length of the amplified DNA molecules usually shows two alternative copies of the DNA fragment, one for each of the chromosomes on which that sequence resides. The application of the two techniques has varied somewhat in human genetic studies; each has advantages and limitations. Sites of short sequence-length variation have been found to be widely distributed along the chromosomes, making them the most widely used sites in genetic-linkage studies designed to track medically important genes in families.