Polymerase chain reaction fragments, detection

Schwartz, H. E., Ulfelder, K., Sunzeri, F. J., Busch, M. R, and Brownlee, R. G., Analysis of DNA restriction fragments and polymerase chain reaction products towards detection of the AIDS (HIV-1) virus in blood, /. Chromatogr., 559, 267, 1991. 

Sheffield, V.C., Cox, D.R., Lerman, L.S. and Myers, R.M. (1989) Attachment of a 40-base pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by polymerase chain reaction results in improved detection of single-base changes. Proceedings of the National Academy of Sciences USA 86, 232—236. 

Fig. 5.2.6 Long-distance polymerase chain reaction (PCR) to detect large deletions and insertions in the low-density lipoprotein receptor (LDLR). The structure of the LDLR gene is shown from exon 1 through 18. The five fragments produced by the five long-distance PCRs are outlined. PCR1 covers exons 1-5, PCR2 exons 6-13, PCR3 exons 15-18, PCR 4 exons 2-10, and PCR5 exons 12-18...

Charbonnier F, Raux G, Wang Q, Drouot N, Cordier F, Limacher JM, Saurin JC, Puisieux A, Olschwang S, Frebourg T. Detection of exon deletions and duplications of the mismatch repair genes in hereditary nonpolyposis colorectal cancer families using multiplex polymerase chain reaction of short fluorescent fragments. Cancer Res 2000 60(ll) 2760-2763. 

The excellent resolving power of gel or polymer solution-filled narrow-bore channels readily provides sequencing-grade separation of very similar size DNA molecules, usually detected by laser-induced fluorescence [9]. Collection of any individual or all separated fragments by capillary electrophoresis (CE) proved to be feasible and provided enough material for such downstream processes as polymerase chain reaction (PCR), sequencing, or cloning [10-12],... 

Polymerase chain reaction is particularly useful for genetic analysis because both amplification and primer specific isolation of gene fragments occur simultaneously. Allele-specific amplification can be employed to detect a single base-pair mutation through the use of a specially designed primer which is complementary to... 

Detection with a microchip is primarily through LIF, since this is eashy implemented with the planar configuration of the microchip (Figure 5-10). Limits of detection for fluorescein-like fluors have been easily demonstrated at the 10 M level and pushed as low as 10 M—a mass detection limit of a few hundred molecules. This allows for detection, for example, of polymerase chain reaction (PCR)-amplified DNA fragments at a level that competes with P-autoradiography jffom Southern blots. Typical microchip separation times are around 50 to 200 seconds. 

Detection of EHEC-associated markers is performed either directly from the whole genome (DNA-hybridization) or indirectly after amplification by Polymerase Chain Reaction (PCR) of specific gene fragments. 

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