ISSN:0173-0835    

DOI:10.1002/elps.1150181102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractStable low molecular weight (LMW) RNA comprises molecules used in the taxonomy of microorganisms and in studies on the microbial diversity of populations. However, the use of electrophoretic techniques has been hampered due to the low resolution obtained with techniques used for the separation of this kind of molecule. In this work we develop an electrophoretic method (staircase electrophoresis) that increases the resolution of the technique. This improvement in the resolution adequately resolves the three zones that integrate the profiles of LMW RNA: ribosomic 5S RNA (5S rRNA), class 2 transfer RNA (tRNA), and class 1 transfer RNA, allowing the technique to be applied to taxonomic studies (diagnostic, the identification of the individuals), phylogenetic studies and studies on naturally occurring microbial populations.

ISSN:0173-0835    

DOI:10.1002/elps.1150181103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractRecently, we found that, in concentrated neutral solutions, DNA molecules migrate in linear conformation under steady electric field. In this paper, we report the conformational change of DNA during 120° crossed‐field electrophoresis in the same polymer solution. We found that, in concentrated polyacrylamide solutions, the reorientation process of DNAs becomes simple: the DNA goes back along the previous track and the reorientation time is longer motion for the separation of DNA fragments in pulsed field gel electrophoresis. We expect that this phenomenon is useful for a more efficient separation technique of large DNAs than the current pulsed field gel electrophores

ISSN:0173-0835    

DOI:10.1002/elps.1150181104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe efficiency of individualisation using nonisotopic chemiluminescent‐ enhanced probes (NICE™) was investigated by analysing DNA fingerprints obtained from 190 unrelated Caucasians. Novel analysis of the scoring procedure enabled us to include the comparison of 585 pairs of samples for each of two probes. When the results of NICE probes 33.6 and 33.15 were combined, the mean percentage band share between two unrealated individuals was 16.8% and the mean number of bands identified in an individual DNA fingerprint was 54.8. Results were compared with those obtained using isotopically labelled probes and suggest that the two labelling systems gave similar efficiencies for differentiating between individuals. Analysis of DNA fingerprints from 37 family trios (mother, child and father groups) gave a mutation rate of 0.10% when using NICE probes. The two labelling systems compared were equally efficient in establishing family relationsh

ISSN:0173-0835    

DOI:10.1002/elps.1150181105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe STR system D12S67 was amplified by polymerase chain reaction (PCR) and analyzed by denaturing gel electrophoresis followed by silver staining. Among 133 DNA samples from Japanese individuals, 11 alleles were observed and the heterozygosity was 80%. When sequences of the alleles were compared, each allelic segment contained 35–45 gata or gaca tetranucleotide repeats. Although a (gaca)nrepeat block was concentrated in a defined region, nine different blocks of (gata)nrepeats were observed separated by the (gaca)nrepeat and single copy sequences. In addition, the allelic differences result from the total number of repeats of at least three (gata)nand the (gaca)nrepeat regions. Novel primers overlapping part of the sequence with each other were constructed in the center of the D12S67 sequence, and the 5′ and 3′ segments were amplified by PCR. Both of these segments were highly polymorphic and showed heterozygosities of 77 and 78% with 7 and 10 alleles, respectively. The genotypes of the two polymorphisms were not concordant with the original polymorphism and the combination of the 5′ and 3′ side segment polymorphisms enabled further detailed classification of the D12S67 locus by simple comparison of the PCR product sizes. The number of the allele types increased up to 35 and the heterozygosi

ISSN:0173-0835    

DOI:10.1002/elps.1150181106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractDetection on automated DNA sequencers of polymerase chain reaction (PCR) products of tetra‐ and penta‐nucleotide short tandem repeat (STR) loci frequently reveals one or more extra peaks along with the true, major allele peak. The most frequent extra peak pattern is a single smaller peak which is one repeat unit shorter than the true allele peak. The existence of such artificial peaks is of special importance when the methods are used for forensic investigations because the artificial extra peaks may simulate true alleles when samples containing mixtures of DNA from different individuals are analyzed. We have investigated the relative levels of formation of extra peaks in 14 STR marker systems. We found that not only the parameters of the PCR but also factors determining the stringency during the post‐PCR and pre‐electrophoresis handling of samples were of importance for the formation of extra peaks. In our hands, the amounts of extra peaks were reduced (i) if the samples were effectively denatured immediately before loading, (ii) if they contained substantial amounts of formamide (i.e.>50%), and (iii) if the temperature of the electrophoresis gel was above a certain level (i.e.>43°C). The results suggest that extra peaks may in part be due to re‐annealing of the PCR product under suboptimal conditions. When efforts had been made to reduce the post‐PCR formation of extra peaks, the relative peak areas of the extra peaks ranged from 1% to 17% of those of the true alleles. Similar results were obtained when the PCR products were analyzed under native conditions. Low‐copy genome analysis excluded that somatic heterogeneity of the STR regions caused the extra peaks. The systems HumVWA31A, HumFibra/FGA, and D21S11 were especially affected by low‐stringency conditions, while Hum‐TH01, HumCD4, and D12S391 were virtually unaffected by low‐stringency conditions. Replacement of theTaqDNA polymerase with DNA polymerases with lower processivity resulted in higher levels of extra peaks. Our results support the hypothesis that extra peaks are produced due to slipp

ISSN:0173-0835    

DOI:10.1002/elps.1150181107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe fifth allele,DNASE1*5, of human deoxyribonuclease I (DNase I) has been discovered. Polymerase chain reaction fragments containing exon 5 of the DNase I gene were screened for DNA polymorphism using single‐strand conformation polymorphism (SSCP) analysis. DNAs from 114 unrelated Japanese and 81 German individuals were tested and a new variant was detected. By DNA sequencing analysis, this variant was found to be caused by a heterozygous G‐A transition at nucleotide position 1227 that results in a Val to Met substitution at amino acid position 92 of the mature enzyme. The nucleotide substitution was also confirmed by mismatched polymerase chain reaction (PCR)‐restriction fragment length polymorphism (RFLP) analysis. Genotyping of the variant could be carried out by three independent reactions based on PCR amplification, and phenotyping by isoelectric focusing followed by immunostaining. The results supported the presence of the fifth codominant allele,DNASE1*5, which generates a new is

ISSN:0173-0835    

DOI:10.1002/elps.1150181108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe genotyping of mitochondrial acetaldehyde dehydrogenase (ALDH2) is very important in alcohol studies. We describe an ALDH2 genotyping method based on nonradioactive single‐strand conformation polymorphism (SSCP) analysis on mini‐gels following amplification with a mutated primer set. The three ALDH2 genotypes were clearly and unambiguously distinguished. This method was applied to the ALDH2 genotyping of 129 unrelated Japanese. The allele frequency of ALDH2*2 was calculated to be 0.271, which was consistent with the previous data. The method proved to be simple, rapid and reliable, and dispensed with isotopic reagent and expensive restriction enzymes and equipment. The SSCP method described here is valuable in routine ALDH2 genotyp

ISSN:0173-0835    

DOI:10.1002/elps.1150181109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractA T‐deletion at position 10 of exon 4 for catalase gene was reported as a novel mutation, causing a new genetic type of acatalasemia in Japan. This mutation, destroying aHinf1 recognition site, was searched for in Hungarian acatalasemic (2) and hypocatalasemic (22) patients and in controls (27) byHinf1 digestion and sequence analyses of a 203 bp polymerase chain reaction (PCR) product containing the entire exon 4. TheHinf1 polymorphism did not reveal any difference between controls and hypocatalasemic as well as acatalasemic patients. These results were confirmed by sequence analyses showing the T nucleotide for the two acatalasemic and for one unrelated hypocatalasemic patient, as well as for two controls. These findings represent further evidence that acatalasemia is heterogeneous at the DNA leve

ISSN:0173-0835    

DOI:10.1002/elps.1150181110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractTwo different approaches (stepwise and parallel mechanism) for describing the dissociation of amphoteric protolytes are described. Additionally, the classification of the possible model on the basis of the ampholyte lifetime state is proposed. The possibility of application of different schemes is discussed. It is suggested that the correct description may be based only on highly relaxing models. The formulas describing the properties of ampholyte solutions are derived for the case of two ionogenic groups. It is demonstrated that the incorrect choice of the dissociation model may result in essential mistakes in calculation of such values as buffer capacity, electrophoretic mobility, and conductivity. On the basis of the theory here developed, the interpretation of natural pH‐gradient conductivity is given. Some terminology questions are also discusse

ISSN:0173-0835    

DOI:10.1002/elps.1150181111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY