PCR/ESI

FIGURE 19.4 Workflow of the PCR/ESI-MS process. Step 1 Extraction and amplification of target DNA with coamplification of calibrant DNA for quality control and semiquantitative analysis of PCR products. Step 2 Mass spectrometric analysis is performed separately on each well in a TOE mass spectrometer with ESI. Step 3 Data processing to determine base composition from mass spectrometric signal and correlation of that base composition to a database of known base compositions for each primer pair to determine organism identification [32]. 

The third and final example used to illustrate the capabilities of PCR/ESI-MS in the TIGER study was using alphaviruses. Members of the genus Alphavirus,... 

Additional research has been conducted on a variety of other viruses, including adenoviruses, which cause several types of respiratory infections [34], and influenza [35]. The PCR/ESI-MS technology was at the forefront as a diagnostic method during the recent HlNl epidemic due to its ability to objectively assess strain variation during detection [36]. 

The held of PCR/ESI-MS is currently dominated by Abbott Molecular (Abbott Park, IL) their PLEX-ID platform contains an in-house database that is crucial for data analysis. As a recommended protocol for this type of work, the hrst step is extraction of DNA from the mixture to be analyzed. Many different types of extraction kits exist and are suitable for PCR-MS analysis however, most published studies have used the Qiagen DNA extraction kits (Qiagen, Valencia, CA) and KingFisher extraction (Thermo Scientihc, Waltham, MA). The resulting DNA is then aliquoted into a 96-well plate for PCR, where each well contains a single set of broad-range primers to amplify the DNA present. 

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... 

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. 

Since alterations in global DNA methylation are implicated in various pathobio-logical processes, a gradient IPC-ESI-MS/MS method with a volatile IPR was used to determine cytosine and 5-methylcytosine in DNA quantification relied on stable isotope dilution [58], Muscular dystrophies caused by various mutations in the dystrophin gene are amenable to easier prenatal diagnosis via a multiplex polymerase chain reaction (PCR)/IPC assay [59]. Some guidelines for the analysis of genomic DNA by IPC-ESl-MS can be found in Reference 60. 

Krahmer et al. [48] reported the use of quadrapole ESl-MS and ion-trap ESI-MS-MS for the identification of SNP in the PCR products of the Pro and Arg variants of the tumour suppressor protein p53 gene. The 69-bp Arg variant is isomeric with the Pro variant. A 43-bp fragment, created by means of restriction enzyme digestion, could be sequenced and characterized. 

A.P. Null, L.T. George, D.C. Muddiman, Evaluation of sample preparation techniques for mass measurements of PCR products using ESI-FT-ICR-MS, J. Am. Soc. Mass Spectrom., 13 (2002) 338. 

Genotyping SNP using intact PCR products by ESI-MS, Rapid Commun. Mass Spectrom., 15 (2001) 1752. 

A. P. Null, J.C. Hannis D.C. Muddiman, Preparation of single-stranded PCR products for ESI-MS using the DNA repair enzyme lambda exonuclease. Analyst, 125... 

M.T. Krahmer, Y.A. Johnson, J.J. Walters, K.F. Fox, A. Fox, M. Nagpal, ESI-MS analysis of model oligonucleotides and PCR products determination of base substitutions, nucleotide additions/deletions, and chemical modifications. Anal. Chem., 71 (1999)2893. 

We have developed a method for analysis of genetic alterations in PCR amplified DNA fragments using electrospray ionisation mass spectrometry (ESI-MS) termed Short Oligonucleotide Mass Analysis (SOMA). The technique is highly accurate and has been applied to genotyping individuals and to mutational analysis of human tumours. 

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