PCR Products in the Clinic

For instance, a direct PCR test is sometimes used for genetic diagnosis. An early test for the most common cystic fibrosis (CF) allele (the DF508 mutation) was based on direct measurement of the length of the amplified DNA fragments. Because the mutation involves deletion of three base pairs, a simple test was designed in which primers that flank the missing codon are used for PCR. If no DF508 mutation is present, the PCR fragment will be 100 bases in length. However, the DF508 allele will produce a fragment 97 bases in length. The PCR fragments are separated on a gel and the bands of DNA are stained with a fluorescent dye. The amplified DNA from individuals who do not carry the DF508 mutation will display only the 100 base fragment. Carriers will have one fragment 100 bases in length and a second fragment 97 bases in length. And those homozygous for the DF508 mutation will show only the 97 base PCR fragment. A second type of direct PCR test, such as for the mutation that causes multiple endocrine neoplasia type 2B (MEN2B), involves amplification of a PCR fragment that spans a restriction endonuclease cleavage site that is eliminated by the mutation.

PCR amplification is also used for diagnostic DNA sequence analysis. For instance, the current commercial protocol for detection of mutations in the breast cancer susceptibility genes BRCA1 and BRCA2 begins with PCR amplification of the patient's DNA. The amplified PCR fragments are then subjected to DNA sequence analysis. Because these are very large and complex genes with multiple introns, it takes 35 separate PCR amplifications to span the protein coding region of the BRCA1 gene and 47 reactions to cover the protein coding region of the BRCA2 gene. The interval to be amplified is defined by a different primer pair for every reaction. Both the paternal and maternal chromosomes in the patient's DNA serve as templates for a given amplification. In most cases, the two chromosomes will yield identical fragments. Sometimes the sequences may differ by an inconsequential polymorphism, but in other cases, there may be substantial differences. In the majority of cases, these differences are detected by DNA sequence analysis of the PCR products. However, this is not always the case. For example, a substantial proportion of BRCA1 founder mutations in the Dutch population is caused by deletions of part of the BRCA1 gene. The missing DNA cannot be amplified into a PCR product, and therefore only the normal BRCA1 gene on the patient's other chromosome can be detected. The amplification of only one fragment cannot be distinguished from the amplification of identical paternal and maternal fragments, so the deletion mutation is not detected.

Still other diagnostic protocols are based on hybridization of the PCR fragments representing the two parental chromosomes with each other. The locations at which the sequences do not match can be pinpointed because the mismatches are vulnerable to cleavage by enzymes or chemicals, and the resulting fragment sizes can be measured. Alternatively, amplified PCR fragments can be hybridized to synthetic nucleotide sequences that are anchored on a substrate. The hybridization and washing conditions are adjusted so that only sequences that match exactly will remain stable hybrids at the end of the procedure.

This discussion should convey an impression of the diverse uses of the polymerase chain reaction for genetic diagnosis. Because of its versatility, ease of use, and low cost, PCR is truly the workhorse of molecular genetic analysis. An awareness of how the reaction works is necessary for an appreciation of its tremendous strengths and its limited shortcomings, both of which influence the interpretation of the genetic tests that are based on this technological innovation.

Summary: PCR amplification lends itself to a variety of uses in diagnostic testing, including direct use of PCR products, DNA sequence analysis, and hybridization protocols.

Clinical Significance: Use of PCR amplified fragments can streamline genetic testing procedures, but also introduces limitations. If a mutation, such as a deletion or rearrangement, prevents amplification, a mutation may go undetected.

Glossary Terms for this Page of the Courseware

Polymerase Chain Reaction (PCR)
The technique of PCR permits the selective amplification of a specific segment of DNA or RNA. Some sequence information must be known prior to PCR so that primers flanking the segment of interest can be synthesized. These primers set the boundaries of the segment to be amplified. PCR involves successive cycles of amplification, each of which results in a doubling of the amount of targeted nucleic acid sequence. The process results in millions of amplified DNA fragments representing the targeted sequence.(Back to text)

Cystic Fibrosis
Cystic Fibrosis (CF) is an autosomal recessive genetic condition caused by mutations in the cystic fibrosis transmembrane regulator protein (CFTR). Because CFTR is involved in chloride transport, salt balance is disturbed in affected individuals. The resulting thick mucus causes recurrent lung infections. Intestinal and pancreatic duct blockages are also common.(Back to text)

Multiple Endocrine Neoplasia Type 2B (MEN2B)
MEN2B is a cancer syndrome that results from a single mutation in the RET gene. This syndrome is characterized by skeletal abnormalities and neuromas, in addition to medullary thyroid carcinoma and pheochromocytoma. (Back to text)

Deletion
A deletion is a mutational event in which a segment of DNA is removed from the previous sequence of bases. The size of a deletion can range from a single base or nucleotide to a major part of a chromosome. The effects of a deletion may range from undetectable to highly deleterious, depending on the size of the deletion and the importance of the region to the development and function of the organism. (Back to text)

Hybridization
When single-stranded nucleic acids are incubated under conditions so that only complementary hybrids can form, the process is referred to as hybridization. (Back to text)

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