Evaluation of T-cell receptor gene rearrangements in patients with recurrent patch/plaque (T2) CTCL (mycosis fungoides)

Cutaneous T-cell lymphoma is typically a clonal neoplasm of epidermotropic CD4+ T-lymphocytes that includes the entity mycosis fungoides (MF). After identification of patients with recurrent MF treated with total skin electron beam therapy (TSEBT) at the Yale University School of Medicine, this study attempted to compare T-cell receptor (TCR) gamma gene rearrangements via polymerase chain reaction (PCR) in both original and recurrent skin biopsies from these patients. Between 1974 and 1996, a total of 95 T2 MF patients were treated with TSEB, and four of these were identified for the study. Slides and tissue samples of both primary and recurrent skin biopsies for each patient were confirmed as being consistent with ME DNA for PCR was isolated from paraffin-embedded tissue samples. Using consensus primers that hybridize with conserved regions of the TCR gene, these regions of the genome were amplified. The PCR products were then analyzed by acrylamide gel electrophoresis. Of the primary and recurrent samples from four patients with a median disease-free interval (DFI) of 1222 days, only two showed evidence of a dominant TCR clone. A number of factors, including lack of sequence homology between the primers and the gene segments, the existence of multiple neoplastic cell lines, DNA degradation in the archival samples, and the presence of reactive as well as malignant lymphocytes, may have prevented the detection of dominant TCR rearranged clones in the samples. Despite the results of this study, TCR analysis via PCR and gel electrophoresis continues to be of utility in the evaluation of patients with MF when used in conjunction with other diagnostic modalities and in cases with nonspecific clinical, histopathological, and immunophenotyping findings.     

Molecular nature of beta-thalassemia in Tajikistan: a four base pair deletion in codons 41-42 of the beta-globin gene]

Thirty tajiks, whose relatives had beta-thalassemia traits (revealed in previous investigations by determination of the HbA-2 and HbF levels) were selected to screen beta-thalassemia mutations. DNA samples from each individual were subjected to the PCR (polymerase chain reaction) to amplify the 635 bp beta-globin gene fragment. One additional band was detected in three samples after the amplified fragment underwent electrophoresis in 2% agarose gel and the EtBr was stained, and two additional ones were revealed by 6% PAAGE and staining of the EtBr. All additional bands migrated more slowly than appropriate 635 bp fragment. It is supposed that additional bands are heteroduplexes formed from the wild type chains and mutated chains carrying a deletion or insertion. The 4 bp deletion of the 41-42 (-tctt) was detected after the direct sequencing of the amplified fragments. This mutation is common among Chinese but it was not revealed in the Middle Asia populations. The mutation can be easily screened using the PCR and electrophoresis in 2% agarose gel or PAAG of the amplified beta-globin gene fragments.     

Two DNA sequences specific for the canine Y chromosome

Data are presented on the characterization of two nucleotide sequences found exclusively in the DNA of male dogs. In polymerase chain reactions (PCRs) of canine genomic DNA with a decanucleotide primer of arbitrary sequence (OP-W17), two nucleotide segments (650 and 990 bp) were amplified only from male samples, whereas a number of other fragments between 400 and 2500 bp in size were amplified from both male and female samples. The two male-specific segments were cloned and sequenced, and terminal 24mer oligonucleotide primer pairs were synthesized. PCR with these specific primer pairs resulted in amplification of the two male-specific sequences only from DNA samples of 34 male dogs; no product was amplified from 42 samples of females. A segment of the SRY gene previously localized on the Y chromosome could be amplified in DNA samples that had the two new sequences. Eco RI digested genomic male DNA when hybridized with the 650 bp or the 990 bp sequences, resulted in a single band for each on Southern analysis; DNA from females did not yield any bands. Comparisons between the two new sequences and the SRY gene segment revealed no homologies. We concluded that the two new sequences are specific for the canine Y chromosome and do not contain the short characterized segment of the SRY gene.     

儿童急性淋巴细胞白血病特异性标记物的精准检测方法研究

目的：T细胞和免疫球蛋白重链基因重排是微小残留病灶水平的特异性标记物,而微小残留病灶的水平与儿童急性淋巴细胞白血病的复发强烈相关。应用传统的聚合酶链式反应方法来监测IgH／TCR基因重排不仅耗时、耗人力,而且敏感度较低。本研究旨在探索一种更为高效和敏感与实用的监测IgH／TCR基因重排的精准检测方法。方法：应用多重PCR技术检测26个患有急性淋巴细胞白血病的儿童的外周血样品中的标记物,这些儿童是在中国哈尔滨市最近两年内被诊断的患者。分别应用基因扫描和毛细血管电泳方法检测IgH（FRI,FRII,FRⅢ）／TCR（TCRB,TCRγ）基因重排和分析PCR产物的片段。结果：IgH／TCR基因重排和对IgH基因重排的阳性率分别为92．3％和75％,在26个病例中,4个复发病人的IgH的三个片段（FRI,FRII,FRⅢ）基因重排显示阳性。进一步分析显示复发与ign基因重排呈线性相关。结论：实验与临床应用表明,基因扫描这种方法对于IgH／TCR基因重排的检测是可靠的、实用的,因而可用于儿童急性淋巴细胞白血病的诊断和随访。     

Quantitative analysis of DNA aberrations amplified by competitive polymerase chain reaction using capillary electrophoresis

We compared the use of capillary electrophoresis (CE) in a polymer network with the use of slab gel electrophoresis for the quantitative analysis of polymerase chain reaction (PCR)-amplified DNA samples. We quantified residual lymphoma cells carrying a translocation between chromosomes 14 and 18, in consecutive patient peripheral blood samples that were amplified by competitive PCR. For CE analysis we used a 4% linear polyacrylamide network. Results show that the calculated number of translocations in patient samples using both analyses were comparable. We conclude that CE is a sensitive, non-radioactive, fast and accurate method for quantitation of competitive PCR products.     

Detection of Streptococcus pneumoniae DNA by using polymerase chain reaction and microwell hybridization with Europium-labelled probes

The present paper describes a novel modification of polymerase chain reaction (PCR) for the detection of Streptococcus pneumoniae DNA in clinical specimens. PCR was based on the detection of a 209-base pair segment of the S. pneumoniae pneumolysin gene. For the demonstration of the amplification product, microwell hybridization with a Europium-labelled oligonucleotide probe complementary to a biotinylated strand of the PCR product was performed, and the presence of the PCR product was monitored by time-resolved fluorescence (TRF) of the Europium chelate. The sensitivity of the assay for purified S. pneumoniae DNA was 50 fg DNA corresponding to 20 genome equivalents of S. pneumoniae DNA. The efficiency of the hybridization step was monitored by using known amounts of synthetic target oligonucleotides as standards. Sensitivity of 3×10⁸ molecules per individual reaction well was achieved with a 30-min attachment time and a 3-h hybridization time.

Detection of PCR-amplified products by the microwell hybridization technique and TRF was compared to agarose gel electrophoresis in 50 middle ear fluid samples obtained from children with acute otitis media. The agarose gel and TRF detection methods identified all culture-positive samples, but both were also positive for 55% of the culture-negative samples. The results suggest that the detection of amplified PCR products by microwell hybridization using Europium-labelled oligonucleotides is a reliable method for the demonstration of the pneumolysin gene fragment. Furthermore, the method is suitable for automation and, thus, for testing high numbers of samples. The clinical significance of the PCR findings remains to be studied.     

Retrieval of glycoside hydrolase family 9 cellulase genes from environmental DNA by metagenomic gene specific multi-primer PCR

A new method, termed metagenomic gene specific multi-primer PCR (MGSM-PCR), is presented that uses multiple gene specific primers derived from an isolated gene from a constructed metagenomic library rather than degenerate primers designed based on a known enzyme family. The utility of MGSM-PCR was shown by applying it to search for homologues of the glycoside hydrolase family 9 cellulase in metagenomic DNA. The success of the multiplex PCR was verified by visualizing products on an agarose gel following gel electrophoresis. A total of 127 homologous genes were amplified with combinatorial multi-primer reactions from 34 soil DNA samples. Multiple alignments revealed extensive sequence diversity among these captured sequences with sequence identity varying from 26 to 99.7%. These results indicated that significantly diverse homologous genes were indeed readily accessible when using multiple metagenomic gene specific primers.     

Amplification of PFGE-separated chromosome-specific DNA by degenerate oligonucleotide-primed PCR (DOP-PCR)

Specific probes for DNA molecules of high molecular weight can be efficiently and rapidly prepared by the combination of pulsed-field gel electrophoresis (PFGE) with degenerate oligonucleotide-primed PCR (DOP-PCR). More than 10 mg of a specific probe were amplified from only 1 ng of a PFGE-separated DNA fragment of a red alga, Cyanidioschyzon merolae. The probe hybridized specifically to the fragment which was used as a template for DOP-PCR.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

正常人外周血αβ T细胞TCR β链CDR3多态性和长度分析

采用免疫扫描谱型分析技术,分析正常人外周血T细胞TCR β链CDR3的多态性和长度分布.提取6例正常人外周血单个核细胞（peripheral blood mononuclear cell PBMC）的总RNA,逆转录成cDNA,以24个TCR BV基因家族为上游引物,共同的TCR BC基因为下游引物（荧光标记）,6例样品PCR产物的基因扫描分析表明,24个TCR BV家族的CDR3谱型分析在1.5%琼脂糖凝胶电泳图上显示1个模糊条带.序列测定结果显示,各TCR BV家族的CDR3谱型均超过8个条带.GeneScan分析证明,正常人外周血T细胞的24个TCR BV家族的CDR3谱型为标准的高斯分布,各家族的CDR3表达频率相近,呈现不同的CDR3多态性和不同的CDR3长度.多数TCR BV家族产物为框架内重排（相邻2个PCR产物相差3个碱基）,少数TCR BV家族表现为两个峰群的重排.该研究对正常人TCR β链CDR3谱系的详细分析将为T细胞应答、TCR重组等研究提供基础.利用免疫谱型分析技术能监测到TCR CDR3谱型分布特征和表达频率的变化.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60 degrees C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

Automated DNA mutation analysis by single-strand conformation polymorphism using capillary electrophoresis with laser-induced fluorescence detection

Automation is essential for rapid genetic-based mutation analysis in clinical laboratory to screen a large number of DNA samples. We propose in this report an automatic process using Beckman Coulter P/ACE™ capillary electrophoresis (CE) with laser-induced fluorescence (LIF) system to detect a single-point mutation in the codon 12 of human K-ras gene. Polymerase chain reaction (PCR) using a fluorescently labeled reverse primer and a plain forward primer to specifically amplify a selected 50 bp DNA fragment in human K-ras gene. The amplified DNA is placed on the sample tray of the CE system with a pre-programmed step for single-strand conformation polymorphism (SSCP) analysis. Sample injection and denaturation processes are performed online along with separation and real-time data analysis. The concept of automation for rapid DNA mutation analysis using CE-LIF system for SSCP is presented.     

Denaturing gradient gel electrophoresis detection of polymorphism in a PCR fragment of sheep epidermal growth factor gene

The ovine map is not yet well-developed, which represents a problem when looking for markers of a region of interest in sheep. A means of circumventing this is to use comparative mapping. In this study primers were determined using consensus sequences for the epidermal growth factor gene of humans, rats and mice, and an ovine epidermal growth factor gene fragment was amplified by polymerase chain reaction (PCR). A new set of specific ovine primers was chosen to study the polymorphism of this DNA fragment by denaturing gradient gel electrophoresis. Eighty-four individuals belonging to seven sheep breeds were studied with this technique and four alleles were detected. The heterozygosity rate was 0.57. Family analysis showed mendelian inheritance of the alleles. Usually, genetic analysis of type-I loci used in the comparative mapping is based on the detection of restriction fragment length polymorphisms in sheep DNA using cDNA probes from other species. Our work shows that another method, based on PCR and denaturing gradient gel electrophoresis techniques, can be efficiently used.     

Identification of a RAPD marker linked to progressive rod-cone degeneration in dogs

Random amplified polymorphic DNA (RAPD) analysis has been used widely in plant and fungi for identification of markers linked to genetic traits and mapping, but its use is limited to identification of intra- and inter-species difference in domestic mammals. We report here identification of a RAPD-derived marker linked to progressive rod-cone degeneration (prcd), an inherited autosomal recessive retinal disease of dogs. A total of 400 standard 10-mer primers were used for amplification by use of DNA samples from normal (+/+) and affected (prcd/prcd) dogs. A single primer was identified which amplified a 1.5-kb DNA fragment only from normal dogs. PCR with longer primers designed from the sequence-characterized amplified region of the 1.5-kb DNA fragment identified a co-dominant multi-allelic polymorphism in the prcd-informative pedigree. Three recombinants were identified among 34 informative offsprings, yielding a LOD score of 5.568 at θ= 0.091. This marker was mapped to two canine–rodent hybrid cell lines in which two genes (canine homologues of human breast cancer 1 susceptibility gene, and cGMP phosphodiesterase γ-subunit gene), and three anonymous microsatellites have been identified. This is the first reported identification of a RAPD-derived marker with multiple alleles linked to a mammalian disease locus. Received: 20 February 1998 / Accepted: 24 April 1998     

Improved Identification of Rapidly Growing Mycobacteria by a 16S–23S Internal Transcribed Spacer Region PCR and Capillary Gel Electrophoresis

The identification of rapidly growing mycobacteria (RGM) remains problematic because of evolving taxonomy, limitations of current phenotypic methods and absence of a universal gene target for reliable speciation. This study evaluated a novel method of identification of RGM by amplification of the mycobacterial 16S–23S rRNA internal transcribed spacer (ITS) followed by resolution of amplified fragments by capillary gel electrophoresis (CGE). Nineteen American Type Culture Collection (ATCC) Mycobacterium strains and 178 clinical isolates of RGM (12 species) were studied. All RGM ATCC strains generated unique electropherograms with no overlap with slowly growing mycobacteria species, including M. tuberculosis. A total of 47 electropherograms for the 178 clinical isolates were observed allowing the speciation of 175/178 (98.3%) isolates, including the differentiation of the closely related species, M. massiliense (M. abscessus subspecies bolletii) and M. abscessus (M. abscessus sensu stricto). ITS fragment size ranged from 332 to 534 bp and 33.7% of clinical isolates generated electropherograms with two distinct peaks, while the remainder where characterized with a single peak. Unique peaks (fragment lengths) were identified for 11/12 (92%) RGM species with only M. moriokaense having an indistinguishable electropherogram from a rarely encountered CGE subtype of M. fortuitum. We conclude that amplification of the 16S–23S ITS gene region followed by resolution of fragments by CGE is a simple, rapid, accurate and reproducible method for species identification and characterization of the RGM.     

A sensitive and specific multiplex PCR approach for sex identification of ursine and tremarctine bears suitable for non‐invasive samples

We report a new approach for molecular sex identification of extant Ursinae and Tremarctinae bears. Two Y‐specific fragments (SMCY and 318.2) and one X‐specific fragment (ZFX) are amplified in a multiplex PCR, yielding a double test for male‐specific amplification and an internal positive control. The primers were designed and tested to be bear‐specific, thereby minimizing the risk of cross‐amplification in other species including humans. The high sensitivity and small amplicon sizes (100, 124, 160 base pairs) facilitate analysis of non‐invasively obtained DNA material. DNA from tissue and blood as well as from 30 non‐invasively collected hair and faeces yielded clear and easily interpretable results. The fragments were detected both by standard gel electrophoresis and automated capillary electrophoresis.     

Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer

The transfer of naked DNA is gaining growing acceptance for nonviral gene therapy. Integrity and stability of the DNA used in nonviral gene therapy is known to be decisive for efficacy of gene transfer and transgene expression. Thus, preclinical and clinical studies require the safe storage of DNA preparations to ensure defined quality and conformation. To evaluate the influence of potentially destructive processes on plasmid DNA associated with long-term storage, capillary gel electrophoresis (CGE) analysis of the LacZ-expressing pCMVbeta plasmid over a period of 13 months was performed. The CGE analysis revealed that stable storage conditions at -80 degrees C prevent an increase in open circular (oc) plasmid, preserving the covalently closed circular (ccc) form, which is sought for efficient gene transfer. By contrast, long-term storage of plasmid DNA at 4 degrees C leads to the rapid decline of the ccc form and the increase of oc and linear DNA molecules. The use of naked DNA stored for 1, 2, or 13 months at -80 degrees C showed similar in vivo transfer efficiencies by jet-injection. Therefore, analysis of plasmids by CGE allows the reliable determination of integrity and distribution of the topology of the DNA by quantitative means.     

比格犬MC4R基因多态性与体重相关性的研究

为了分析比格犬黑素皮质素受体-4基因多态性与犬体重的关系, 根据犬MC4R基因DNA外显子序列, 设计MC4R基因特异PCR引物1对, 犬DNA经PCR扩增, 克隆和测序, 寻找和确定犬MC4R基因的多态性位点, 分析多态性与犬体重的关系。结果在比格犬MC4R基因中发现2处单碱基缺失突变, 1个单碱基颠换变异, 存在Psh AⅠ酶切位点, 并基于PshAⅠ酶切位点建立了PCR-RFLP技术。统计分析显示犬MC4R基因型与体重显著相关, 可以考虑将MC4R基因作为犬体重的候选基因。     

Identification of the Hind III polymorphic site in the PAI-1 gene: analysis of the PAI-1 Hind III polymorphism by PCR

The identification of the Hind III polymorphic site in the 3' end of the plasminogen activator inhibitor 1 (PAI-1) gene and a simple method to identify the Hind III polymorphism rapidly in the PAI-1 gene using PCR is described. The Hind III restriction site was identified by restriction site mapping and sequence analysis from a cosmid DNA clone. Genomic DNA was isolated from individual human umbilical cords and a 754-bp fragment of the human PAI-1 gene was amplified by PCR. Aliquots of the PCR products were digested with Hind III and analyzed by agarose gel electrophoresis. The presence of two fragments, 754 and 567 bp, was identified, and they were designated as 1/1 (750-bp band), 1/2 (754- and 567-bp bands), and 2/2 (567-bp band). The PCR method is considerably less time consuming than the conventional DNA genotyping using Southern blot analysis. To ensure that this new method identified the same PAI-1 genotypes as previously identified by Hind III restriction fragment length polymorphism (RFLP), samples were simultaneously genotyped by PCR and Southern blot analysis. Both methods identified the same Hind III genotypes in all the samples, confirming the reliability of this new PCR method for the rapid identification of the Hind III polymorphism in the human PAI-1 gene.