PCR methods and applications最新文献

A simple procedure for direct sequencing of double-stranded PCR products by the dideoxy-termination method has been developed using biotinylated sequencing primers. Sequences of the p53 gene have been obtained from DNA extracted from frozen and formalin-fixed paraffin-embedded cancer tissues. Detection of sequencing ladders was done with chemiluminescent or colorimetric techniques. Both are highly sensitive, but colorimetric detection is less prone to diffusion artifacts, which are common in G-rich regions. Use of this nonradioactive PCR-sequencing protocol allowed rapid and simple determination of p53 gene alterations in human tumors.

In this study we examine the factors that lead to nonspecific DNA synthesis during in situ PCR and solution-phase PCR. It was shown that primer-independent DNA synthesis can produce an intense signal during in situ PCR. This primer-independent pathway was apparently the result of the repair of DNA gaps induced by the heat treatment of the paraffin embedded tissue sections. This non-specific signal could be eliminated by blocking gap repair with dideoxy-TTP, avoiding heat treatment, or DNase pretreatment. The primer-independent signal was also influenced by the length and mode of fixation and the sample tissue itself. Elimination of the primer-independent signal and the use of viral primers in tissues that did not contain the virus showed that nonspecific DNA synthesis could be eliminated by the hot start modification. Primer oligomerization did not produce a signal during in situ PCR, even when it occurred robustly in the amplifying solution. Generation of the primer-independent signal in solution-phase PCR with purified DNA required a cross-linking fixative, heating, the addition of bovine serum albumin, and intact protein-DNA cross-links.

The human gene coding for the apoferritin H subunit belongs to a complex multigene family constituted by the expressed gene and by an undefined number of pseudogenes. We have used a strategy based on PCR to amplify specifically the H pseudogenes from a sample of human genomic DNA. With this approach, three new H pseudogenes have been cloned and characterized by DNA sequence analysis. In addition, we have identified a new type of pseudogene, the size of which (700 bp) is caused by multiple detection events in the putative coding region.

We have studied the effect of repeated DNA sequence, especially Alu repeats, on PCR. Alu repeats are sequences that are approximately 300 bp long and interspersed at a very high copy number throughout the human genome. We amplified part of the human low-density lipoprotein receptor gene containing two Alu repeat sequences in the same orientation, approximately 7.8 kb apart, with unique sequence primers outside these repeats. The major PCR product was a DNA fragment with an in vitro deletion between the Alu repeats. The formation of this product depended on the template concentration and the type of polymerase used. Such a product arose apparently as a result of a "jumping reaction" involving a primer whose extension was terminated prematurely within one Alu repeated followed by annealing of such an incompletely extended primer to the other, distant Alu repeat. No such jumping products were seen when a 0.8-kb region containing two nearby inverted Alu repeats within the human alpha-galactosidase A gene was subject to PCR with unique sequence primers annealing just outside these repeats.

Vectorette PCR permits the specific amplification of DNA segments flanking a known DNA sequence. It enables the application of the PCR where sequence information is only available for one primer site. We now show that vectorette PCR can be used for the systematic mapping and retrieval of transgene flanking DNA. We also show that the sequence of large vectorette PCR fragments can be obtained without cloning, by the production of subvectorette fragments.

The production of informative random amplified polymorphic DNA (RAPD) markers using PCR and a single primer is often accompanied by the generation of artifactual (noninformative) bands as well. When RAPD data are used to compute genetic similarity coefficients, these artifacts (false positives, false negatives, or both) can cause large biases in the numerical values of the coefficients. As a result, some workers have been reluctant to use RAPD markers in the estimation of genetic similarities. Artifactual bands are of two types: those caused by variation in experimental conditions, and those caused by characteristics of the DNA to be amplified. A procedure is described that allows for correction of the bias caused by the first type of artifact, providing that replicate DNA samples have been extracted, amplified, and scored. The resulting data are used to obtain an estimate of the proportion of false-positive and false-negatives bands. These values are then used to correct the bias in the computed similarity coefficients. Two examples are given, one in which bias correction is critical to the results, and one in which it is less important. The maximum percent bias, computed from the estimated proportions of false positives and false negatives in the RAPD data set, is proposed as a criterion for determining whether bias correction of the similarity coefficients is required or not. Although all reasonable efforts should be made to optimize PCR protocols to eliminate artifactual bands, when this is not possible, the methods described allow RAPD markers to compute genetic similarities reliably and accurately, even when artifactual bands resulting from variation in experimental conditions are present.