RFLP analysis: The presence of abnormal genes for PAH can be shown using DNA polymorphisms as markers to distinguish between normal and mutant genes. For example, Figure 34.19 shows a typical pattern obtained when DNA from members of an affected family is cleaved with an appropriate restriction enzyme and subjected to electrophoresis. The vertical arrows represent the cleavage sites for the restriction enzyme used. The presence of a polymorphic site creates fragment “b” in the autoradiogram (after hybridization with a labeled PAH-cDNA probe), whereas the absence of this site yields only fragment “a.” Note that subject II-2 demonstrates that the polymorphism, as shown by the presence of fragment “b,” is associated with the mutant gene. Therefore, in this particular family, the appearance of fragment “b” corresponds to the presence of a polymorphic site that marks the abnormal gene for PAH. The absence of fragment “b” corresponds to having only the normal gene. In Figure 34.19, examination of fetal DNA shows that the fetus inherited two abnormal genes from the parents and, therefore, has PKU.
c.
Value of DNA testing: DNA-based testing is useful not only in determining if an unborn fetus is affected by PKU but also in detecting unaffected carriers of the mutated gene to aid in family planning. [Note: PKU is treatable by dietary restriction of phenylalanine. Early diagnosis and treatment are essential in preventing severe neurologic damage in affected individuals.]
VII. POLYMERASE CHAIN REACTION
PCR is an in vitro method for amplifying a selected DNA sequence that does not rely on the biologic (in vivo) cloning method described on p. 483. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. The method can be used to amplify DNA sequences from any source, including viral, bacterial, plant, or animal. The steps in PCR are summarized in Figures 34.20 and 34.21.
A. Procedure
PCR uses DNA pol to repetitively amplify targeted portions of genomic or cDNA. Each cycle of amplification doubles the amount of DNA in the sample, leading to an exponential increase (2n, where n = cycle number) in DNA with repeated cycles of amplification. The amplified DNA products can then be separated by gel electrophoresis, detected by Southern blotting and hybridization, and sequenced.
1.
Constructing primer: It is not necessary to know the nucleotide sequence of the target DNA in the PCR method. However, it is necessary to know the nucleotide sequence of short segments on each side of the target DNA. These stretches, called flanking sequences, bracket the DNA sequence of interest. The nucleotide sequences of the flanking regions are used to construct two, single-stranded oligonucleotides, usually 20– 35 nucleotides long, which are complementary to the respective flanking sequences. The 3′-hydroxyl end of each oligonucleotide points toward the target sequence (see Fig. 34.20). These synthetic oligonucleotides function as primers in PCR.
2.
Denaturing DNA: The target DNA to be amplified is heated to ~95°C to separate the dsDNA into single strands.
3.
Annealing primers: The separated strands are cooled to ~50°C and the two primers (one for each strand) anneal to a complementary sequence on the ssDNA.
4.