PCR methods and applications最新文献

Loss or gain of an entire chromosome and interstitial deletions or amplifications are hallmarks of several hematopoietic neoplasms. These chromosomal anomalies can be identified by conventional cytogenetic analysis of bone marrow aspirates. We have developed a PCR-based assay to detect loss of chromosome 5q31 loci, in the model system of myeloid disorders with the 5q- chromosome (interstitial deletion of 5q), by taking advantage of a highly polymorphic dinucleotide repeat within the interleukin-9 (IL9) gene on 5q31. In a given sample, quantitation of amplification of individual alleles in a Phosphorimager allowed the representation of alleles to be expressed as a ratio of the larger to the smaller allele. Comparison of these ratios in paired DNA samples from Ficoll buoyant and pelletted fractions provides evidence for allele loss. Results presented here demonstrate that this technique of comparison of ratios of isotope incorporation could be expanded to Investigate any deletion or numerical abnormality in hematopoietic tumors.

A new solid-phase primer extension method has been developed for the quantitation of methylation differences and is described here. The method is less cumbersome than Southern blot analysis, expresses the results in a numerical format, can be adapted to a microtitration well format, and thus allows the analysis of a large series of samples. The model gene analyzed here is the calcitonin gene, but the method can be adapted to the analysis of methylation alterations in any area of the genome. The primer extension method clearly differentiated hypermethylated samples from normally methylated samples and a range for normal values could be determined. In quantitation experiments the method showed linearity in a range from 2% to 100% malignant blasts diluted with normal leukocytes.

Random amplified polymorphic DNA (RAPD) markers have been used for many types of genetic analyses, including genome mapping, genotype fingerprinting, phylogeny reconstruction, and measuring genetic similarities. They suffer from one potential limitation, however, because the PCR that is used to produce informative amplification products often produces artifactual products as well. Optimization of PCR protocols to eliminate artifactual bands completely is often too costly or too time-consuming to be practical. Other methods for handling RAPD artifacts, such as deleting inconsistent or faint bands or using only those bands that are reproducible, introduce false negatives into the data. Simply ignoring artifacts and using all bands introduces false positives. When RAPD data are used to compute genetic similarity coefficients, such artifacts can cause significant bias in the estimation. The three coefficients most widely used with RAPD data, the simple matching coefficient, Jaccard's coefficient and Nei and Li's coefficient, differ in the amount of bias produced by a given level of artifactual bands. The simple matching coefficient and Nei and Li's coefficient always exhibit less percent bias than Jaccard's coefficient. For closely related organisms, Nei and Li's coefficient displays less percent bias than the simple matching coefficient. If new DNA samples possessing RAPD markers not present in the previously analyzed samples are added to a study, values of the simple matching coefficient will need to be computed for all samples, not just the new ones. Jaccard's and Nei and Li's coefficients, however, will not need to be recomputed. Furthermore, only Nei and Li's coefficient has a direct biological meaning (it is an estimate of the expected proportion of amplified fragments shared by two samples because they were inherited from a common ancestor). On the basis of these results, Nei and Li's coefficient is recommended for routine computation of genetic similarities using RAPD data, particularly if PCR artifacts are present.

A new test for the diagnosis of Gaucher disease is described. The test is designed to screen large numbers of clinical specimens from high-risk populations. It consists of duplex PCR amplification of genomic DNA followed by hybridization to alkaline phosphatase-conjugated allele-specific oligonucleotide probes (ASOs). High melting temperature PCR primers were used to increase specificity and eliminate the need for a separate annealing step. All hybridization and washing steps were performed at one temperature. Chemiluminescent detection of signals is fast, and results are easily interpreted directly from x-ray films. Currently, the test is being used in our laboratories to screen Ashkenazi Jewish populations in whom Gaucher disease is common.