Optimized PCR labeling in mutational and microsatellite analysis

The behavior of the dielectric properties of gelatin in the frequency range from 10(3) Hz to 10(7) Hz was investigated and compared with that of the globule protein, bovine serum albumin (BSA), desalted gelatin and BSA being used. Dielectric relaxation was observed for both the gelatin and BSA solutions. The relaxation data were fitted well by the Cole-Cole equation; the Cole-Cole parameter (beta) and the relaxation time (tau) were obtained. For the BSA solutions, tau was proportional to the solution viscosity (eta) at 40 degrees C and 25 degrees C, and the values of beta at 40 degrees C were similar to those at 25 degrees C. For gelatin solution, tau was proportional to eta at 40 degrees C, but was not proportional to eta at 25 degrees C. In addition, the values of beta at 25 degrees C were smaller than those at 40 degrees C. These results indicate that the rotation of gelatin and/or polarization of submolecular groups in the coil state greatly contributed to the dielectric relaxation at 40 degrees C; on the other hand, the formation of cross-linking junctions consisting of helix structures would have affected the dielectric relaxation at 25 degrees C.     

Kinetic PCR analysis: real-time monitoring of DNA amplification reactions

We describe a simple, quantitative assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) simultaneously over the course of thermocycling. The video camera detects the accumulation of double-stranded DNA (dsDNA) in each PCR using the increase in the fluorescence of ethidium bromide (EtBr) that results from its binding duplex DNA. The kinetics of fluorescence accumulation during thermocycling are directly related to the starting number of DNA copies. The fewer cycles necessary to produce a detectable fluorescence, the greater the number of target sequences. Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range. This approach also provides a means of determining the effect of different reaction conditions on the efficacy of the amplification and so can provide insight into fundamental PCR processes.     

Sorting out mix-ups. The provenance of tissue sections may be confirmed by PCR using microsatellite markers

Standard identification systems usually ensure that biopsy material is correctly associated with a given patient. Sometimes, as when a tumor is unexpectedly found, the provenance (proof of origin) of a tissue sample may be questioned; the tissue may have been mislabelled or contaminated with tissue from another patient. Techniques used to confirm tissue provenance include comparing either tissue markers of gender or ABO blood groups; however, these methods have weak confirmatory power. Recently, the use of DNA-based polymerase chain reaction (PCR) techniques has been reported. Paired, formalin-fixed, paraffin-embedded, 10 microns tissue sections were selected from 17 patients, 8 of whom had carcinoma, either by dividing a biopsy section, using sequential biopsies, or sequential biopsy and autopsy tissue. The resulting 36 samples were coded before analysis. In two additional cases, 1-mm fragments of tumor from one patient were included in the tissue block of benign tissue from another patient, the tumor fragments were identified on hematoxylin-and-eosin-stained sections, separately scraped off the glass slide, and analyzed. Tissue from two clinical cases, one of suspected mislabelling and one with a suspected carry-over of malignant tissue were also investigated. Short tandem repeat sequences (STR) or microsatellites, are 2-5 base pair repeats that vary in their repeat number between individuals. This variation (polymorphism) can be assessed using a PCR. A panel of markers of 3 STRs; ACPP, INT 2, and CYP 19 (on chromosomes 3, 11, and 15, respectively) were used. DNA was isolated from the samples after xylene deparaffinization and proteinase digestion, and was then amplified in a radioactive PCR using primers selected to give a product size ranging from 136-178 bases. Amplified products were electrophoresed on denaturing polyacrylamide gels, dried, and autoradiographed. DNA segments were successfully extracted from all samples but one, which was fixed in Bouin's fluid. By comparing allele sizes from the panel, all tissue pairs (other than the Bouin's pair) were successfully matched, the 1-mm tumor fragments were correctly assigned, and the two clinical problems were solved. STRs are highly informative and robust markers, well suited to PCR of small portions of tissue sections, and are an effective method to confirm the provenance of benign and malignant biopsy and autopsy material.     

Local genetic structure in red grouse (Lagopus lagopus scoticus): evidence from microsatellite DNA markers

Allelic variation at seven hypervariable tri- and tetranucleotide microsatellite loci was used to determine levels of population differentiation between 14 populations of red grouse (Lagopus lagopus scoticus) in northeast Scotland, UK. Despite the potential for long-distance dispersal in grouse, and a semicontinuous habitat, significant population divergence was observed (mean RST = 0.153; P < 0.01) and an isolation-by-distance effect detected (Mantel test: P < 0.001). Examination of the spatial trend in principal component scores derived from allele frequencies among populations highlighted a barrier to gene flow that was confounding a simple isolation-by-distance effect. This barrier corresponded to an area of unsuitable habitat for grouse associated with a river system that bisected the study area. Mean genetic relatedness was higher for males than for females in all but one of the study populations, suggesting that the territorial behaviour and natal philopatry displayed by cocks have a manifold effect in generating the observed spatial genetic structure. Lower female relatedness values suggest a higher level of female-mediated gene flow, which is sufficient to prevent the loss of genetic variation from within populations and the onset of inbreeding effects. The potential consequences of local subdivision for red grouse populations are discussed.     

Linkage mapping of fifty-eight new rat microsatellite markers

Fifty-eight new anonymous simple sequence repeats (SSR) were generated and mapped to various rat chromosomes. Among them two genes (rat homologs for human cadherin-14 and mouse fibroblast growth factor-related protein) were mapped on Chromosomes (Chrs) 2 and 11 respectively. The majority of markers were generated from a small insert genomic library specific to Chr 11, 13, 14, and 15. Twenty new markers were mapped to Chr 13, which is known to contain a blood pressure quantitative trait locus (QTL). Several approaches to obtain microsatellite markers are described. The protocols and newly generated markers should be useful for ongoing rat genome project.     

Mapping of selective rat chromosome regions using mouse microsatellite markers

Four hundred eighty mouse microsatellite markers distributed in discrete regions on five mouse chromosomes were screened for producing PCR products in the rat. Ninety-eight of these markers or 20% give distinctive PCR products. Among these ninety-eight markers, twenty-three are polymorphic between the inbred hypertensive Dahl salt-sensitive (S) rat strain and several normotensive rat strains of interest. Fourteen of these polymorphic markers have been mapped to the homologous chromosome regions of the rat, and have further been utilized to localize quantitative trait loci (QTL) for blood pressure in the S rat.     

The aspects of psychophysiological analysis of erroneous actions performed by astronauts

The experience of manned space missions points to the fact that cosmonaut-operator plays an important role in providing the efficiency and reliability of the crew-spacecraft system. However, the spaceflight factors can have a negative effect on working capacity of crewmembers and give rise to their operational errors. The paper deals with the methodical approach to analysis of the interrelationship of errors and psychophysiological state of cosmonauts as well as the ways of its realization. Described are the system of indices used for expert assessment of the psychophysiological state, peculiarities of work-rest schedule, and intra- and intergroup interaction. Potentialities of this approach are evidenced by results of the analysis of data from one of the MIR missions.     

Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA

Methylation analysis of individual cytosines in genomic DNA can be determined quantitatively by bisulphite treatment and PCR amplification of the target DNA sequence, followed by restriction enzyme digestion or sequencing. Methylated and unmethylated molecules, however, have different sequences after bisulphite conversion. For some sequences this can result in bias during the PCR amplification leading to an inaccurate estimate of methylation. PCR bias is sequence dependent and often strand-specific. This study presents a simple method for detection and measurement of PCR bias for any set of primers, and investigates parameters for overcoming PCR bias.     

DNA ploidy determination of early molar pregnancies by image analysis: comparison to histologic classification

BACKGROUND: To improve histologic diagnosis of molar pregnancies, updated guidelines have been proposed recently. These guidelines take into account that less developed molar gestations differ from their fully developed counterparts. OBJECTIVE: To test the validity of these criteria by correlating histologic diagnosis with ploidy determination accomplished by means of image analysis. DESIGN: Fifty archival cases of early molar pregnancy were reclassified according to the new criteria. The diagnosis had to be changed from partial to complete hydatidiform mole (PM to CM, respectively) in 9 cases and from CM to PM in 4 cases. DNA image cytometry could be performed in 40 cases (CM, n = 21; PM, n = 19). RESULTS: There was 100% agreement between histologic diagnosis and a diploid or polyploid histogram in CM and 79% agreement between triploidy and PM, when the updated diagnostic criteria were used. This represents an improvement compared with diagnoses made with former criteria. Nevertheless, problems of correct classification remain: In 3 cases classified as PMs, fetal remnants were accompanied by the histologic appearance of a CM. These 3 cases could represent either a true embryonic development in CM or a twin gestation with one normal pregnancy and one mole, or they could belong to a (very rare) third type of mole. All of them show the same risk of persistent trophoblastic disease observed in classic CM. CONCLUSIONS: As the groups at risk for developing persistent trophoblastic disease can be identified by their DNA histograms, ploidy analysis would be desirable in addition to histologic examination.     

Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers

We have developed a vectorette PCR approach to provide an improved method for isolation of microsatellite repeats. The modified procedure relies on PCR amplification using a vectorette-specific primer in combination with one of a panel of anchored dinucleotide repeat primers. The target DNA to be screened for microsatellite sequences can be from YAC, P1, cosmid, bacteriophage or plasmid clones. We have used this technique to isolate novel, polymorphic microsatellite repeats from clones containing the amelogenin gene (AMGX) located on human chromosome Xp22.3.     

DNA analysis of cystic fibrosis in Brazil by direct PCR amplification from Guthrie cards

A 3 bp deletion of codon 508 (phenylalanine) of the cystic fibrosis (CF) gene constitutes the mutation of most CF chromosomes. The frequency of this mutation (referred to as delta F508), varies considerably between populations, ranging from 26% of the CF mutations in Turkey to 88% in Denmark. To determine the frequency of the delta F508 mutation in Brazilian Caucasoid CF patients, we used direct polymerase chain reaction (PCR) amplification of DNA obtained from dried blood spots on Guthrie cards, followed by ethidium bromide staining of gels. Although the overall frequency of the delta F508 mutation was 47% of 380 CF chromosomes from Brazilian Caucasoids born in five different states, significant interstate differences were observed, ranging from a delta F508 frequency of 27% to 53%. While our method could be used to screen patients and their relatives for carrier testing and prenatal diagnosis, the efficacy of screening only for the delta F508 mutation would be low, and would vary from state to state. Screening for a panel of local mutations will be needed to increase the mutation detection rate and optimize genetic counseling.