Polymerase chain reaction specificity

FIGURE 13.21 Polymerase chain reaction (PCR). Oligonucleotides complementary to a given DNA sequence prime the synthesis of only that sequence. Heat-stable Taq DNA polymerase survives many cycles of heating. Theoretically, the amount of the specific primed sequence is doubled in each cycle. 

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

Figure 40-7. The polymerase chain reaction is used to amplify specific gene sequences. Double-stranded DNA is heated to separate it into individual strands. These bind two distinct primers that are directed at specific sequences on opposite strands and that define the segment to be amplified. DNA polymerase extends the primers in each direction and synthesizes two strands complementary to the original two. This cycle is repeated several times, giving an amplified product of defined length and sequence. Note that the two primers are present in excess.

Electrophysiological studies often demonstrate multiple, asymmetric mononeuropathies, usually axonal in type, that cannot be localized to typical sites of entrapment (Keswani et al. 2002). CSF analysis reveals nonspecific abnormalities, such as elevated protein and mild mononuclear pleocytosis. Polymerase chain reaction (PCR) for CMV DNA and nerve or muscle biopsy may provide more specific diagnostic data (Roullet et al. 1994). 

Sakai, Y., Ishihata, K., Nakano, S., Yamada, T., Yano, T., Uchida, K., Nakao, Y., Urisu, A., Adachi, R., Teshima, R., Akiyama, H., Sakai, Y., et ah (2010b). Specific detection of banana residue in processed foods using polymerase chain reaction. /. Agric. Food Chem. 58, 8145-8151. 

Watanabe, T., Akiyama, H., Yamakawa, H., lijima, K., Yamazaki, F., Matsumoto, T., Futo, S., Arakawa, F., Watai, M., and Maitani, T. (2006). A specific qualitative detection method for peanut (Arachis hypogaea) in foods using polymerase chain reaction. /. Food Biochem. 30, 215-233. 

Recent innovations for detecting malaria include DNA or RNA probes by polymerase chain reaction (PCR). These, however, are not widely available for clinical use. A rapid dipstick test (ParaSight F, Becton-Dickinson, Cockeyville, MD) reportedly has a sensitivity of 88% and a specificity of 97%, which is comparable to microscopy. However, ParaSight F can give false-positive results with rheumatoid factor thus microscopy remains the optimal test. 

Due to the low specificity of culture and clinical diagnosis, the multiplex polymerase chain reaction has been employed successfully in diagnosing Haemophilus ducreyi. 

Both target and signal amplification systems have been successfully employed to detect and quantitate specific nucleic acid sequences in clinical specimens. Polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR) are all examples of enzyme-mediated, target amplification strategies that are capable of producing billions of... 

Spierings, G. Ockhuijsen, C. Hofstra, H. Tommassen, J. Polymerase chain reaction for the specific detection of Escherichia colilShigella. Res. Microbiol. 1993, 144,557-564. 

Beckenbach, K., Smith, M.J. and Webster, J.M. (1992) Taxonomic affinities and intra- and inter-specific vartiation in Bursaphelenchus spp. as determined by the polymerase chain reaction. Journal of Nematology 24,140-147. 

Mullis, K.B., Faloona, F., Scharf, S., Saiki, R., Horn, G. and Erlich, H. (1986) Specific enzymatic amplification of DNA in vitro the polymerase chain reaction. Cold Spring Harbor Symposium of Quantitative Biology 51, 263—273. 

Samson-Himmelstjerna, G. von, Woidtke, S., Epe, C. and Schnieder, T. (1997) Species-specific polymerase chain reaction for the differentiation of larvae from Diclyocaulus viviparus and Dictyocaulus eckerti. Veterinary Parasitology 68, 119-126. 

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