PCR products analysis

Gel electrophoresis and, more recently, CGE have been employed principally in molecular biology and biochemical science for the separation of macromolecules such as proteins and nucleic acids. And GCE has been successfully used in oligonucleotide purity analysis, antisense gene therapy, DNA sequencing, PCR product analysis, and DNA forensics. 

Capillary gel electrophoresis has been employed both for molecular biology applications, such as PCR product analysis and antisense gene therapy, and for protein chemistry applications such as protein separations. 

Future applications of CE to PCR product analysis will arise from improvements in CE instrumentation. Advances in miniaturization of CE devices by producing glass chips with etched channels of <1 cm in length... 

Direct PCR product analysis by electrophoresis is also frequently used in the clinical laboratory to query the quality of intermediary steps prior to the assay. (For example, was the nucleic acid isolated successfully How well was it purified Did the amplification work How specific was the PCR And so on). 

The PCR assays traditionally used in the study of hematological malignancies have entailed a two-step procedure comprising an initial amplification reaction, followed by analysis of the PCR products in a separate process, PCR product analysis has generally entailed size fractionation by gel electrophoresis and product visualization by ultraviolet... 

Applications of CGE include separation of ssDNA, dsDNA, RNA and proteins. Sizing of DNA fragments is a major application. This is important for DNA sequencing (section 6.3) as well as after PCR product analysis (section 6.2). SDS-PAGE of proteins (section 3.2.3) is also commonly performed in capillaries. 

Ulfelder, K., Anderson, K., and Schwartz, H. E., Analysis of PCR products and DNA restriction fragments to detect AIDS (HIV-1) virus in blood, poster presentation, HPCE 91, San Diego, CA, 1991. 

Because the templates compete for amplification and, in the case of reverse transcription PCR (RT-PCR), also for reverse transcription, any variable affecting amplification has the same effect on both. Thus, the ratio of PCR products reflects the ratio of the initial amounts of the two templates as demonstrated by the function C/W=C (l+ )"/Wi(l+ )n, where Cand Ware the amounts of competitor and wild-type product, respectively, and C and W are the initial amounts of competitor and wild-type template, respectively, (Clementi etal., 1993). From this linear relationship, it could be concluded that a single concentration of competitor could be sufficient for quantitating unknown amounts of wild-type templates. However, in practice, the precise analysis of two template species in very different amounts has proved difficult and cPCRs using three to four competitor concentrations within the expected range of wild-type template concentrations are usually performed. In a recent study of different standardization concepts in quantitative RT-PCR assays, coamplification on a single concentration of a competitor with wild-type template was comparable to using multiple competitor concentrations and was much easier to perform (Haberhausen et al, 1998). 

Currently PCR and mass spectrometry are performed by two separate instruments. However, there is no reason why PCR followed by simple automated cleanup and mass spectrometry cannot be incorporated into a single integrated instrument. Essentially every configuration of the modern ESI mass spectrometer has been used successfully for the analysis of PCR products, from the highest to the lowest resolution involving. Fourier transform ion cyclotron resonance (FTICR), triple quadrupole, quadrupole-time of flight (Q-TOF), and ion trap.22-24 MS discriminates between two structurally related PCR products by MW difference. Mass accuracy is needed to differentiate the... 

Two variations on the analysis of PCR products by ESI mass spectrometry have emerged (1) direct-injection MS and tandem mass spectrometry (MS-MS) and (2) liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). In the former approach, the sample is cleaned manually, and as noted above, the cleanup is performed as simply and rapidly as possible. In the latter approach, the cleanup is done automatically... 

Because of the nature of PCR the identifications can be made either directly from clinical or environmental samples or after culture. However, because environmental samples contain many bacterial species, among which bacilli are the most common, the design of primers is important to avoid complex mixtures of PCR products. Such mixtures are extremely difficult to analyze by direct MS analysis. Furthermore the sensitivity of the analysis may be compromised if the signal is spread among many components. Successful analysis directly from environmental samples therefore is still a topic for current research. 

The ability to detect small genetic changes becomes more difficult as mass increases. There is further an upper mass range where analysis is impractical. For low-resolution instruments this limit is around a 100 mer. Thus the mass has to be minimized or a high-resolution instrument employed. Alternatively, the smaller the piece of DNA analyzed, the more it chemically resembles a primer or nucleotide monomer thus separation of the two during cleanup is difficult to do. If the primers and nucleotides are not removed, they can provide a massive background on MS analysis or inhibit ionization of the PCR product by preferential ionization. Thus for practical reasons it is extremely difficult to employ a PCR product below a 40 to 50mer for direct ESI MS or ESI MS-MS analysis. 

Analysis of bacterial monomers is performed routinely to detect bacteria at trace levels in environmental samples using GC-MS-MS. PCR is also used routinely by the molecular biology community for detection of clinical infections. MS and MS-MS greatly improve the specificity of analysis of PCR products and provide additional structural information that may be important in apply-... 

Jiang, Y. Hofstadler, S. A. A highly efficient and automated method for purifying and desalting PCR products for analysis by electrospray ionization mass spectrometry. Anal. Biochem. 2003,316,50-57. 

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