Study of differential polymerase chain reaction of C-erbB-2 oncogene amplification in gastric cancer

ＭＥＴＨＯＤＳＣｅｒｂＢ２ｏｎｃｏｇｅｎｅａｍｐｌｉｆｉｃａｔｉｏｎｗａｓｅｘａｍｉｎｅｄｂｙｕｓｉｎｇｄｉｆｅｒｅｎｔｉａｌｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ（ｄＰＣＲ）ｉｎｓｕｒｇｉｃａｌａｎｄｅｎｄｏｓｃｏｐｉｃｓｐｅｃｉｍｅｎ...

Clonal immunoglobulin heavy chain and T-cell receptor γ gene rearrangements in primary gastric lymphoma

AIM:To study the diagnostic value of immunoglobulin heavy chain(IgH)and T-cell receptorγ (TCR-γ)gene monoclonal rearrangements in primary gastric lymphoma(PGL).METHODS:A total of 48 patients with suspected PGL at our hospital were prospectively enrolled in this study from January 2009 to December 2011.The patients were divided into three groups(a PGL group,a gastric linitis plastica group,and a benign gastric ulcer group)based on the pathological results(gastric mucosal specimens obtained by endoscopy or surgery)and follow-up.Endoscopic ultrasonography(EUS)and EUSguided biopsy were performed in all the patients.The tissue specimens were used for histopathological examination and for IgH and TCR-γ gene rearrangement polymerase chain reaction analyses.RESULTS:EUS and EUS-guided biopsy were successfully performed in all 48 patients.In the PGL group(n=21),monoclonal IgH gene rearrangements were detected in 14(66.7%)patients.A positive result for each set of primers was found in 12(57.1%),8(38.1%),and 4(19.0%)cases using FR1/JH,FR2/JH,and FR3/JH primers,respectively.Overall,12(75%)patients with mucosal-associated lymphoid tissue lymphoma(n=16)and 2(40%)patients with diffuse large B-cell lymphoma(n=5)were positive for monoclonal IgH gene rearrangements.No patients in the gastric linitis plastica group(n=17)and only one(10%)patient in the benign gastric ulcer group(n=10)were positive for a monoclonal IgH gene rearrangement.No TCRgene monoclonal rearrangements were detected.The sensitivity of monoclonal IgH gene rearrangements was 66.7%for a PGL diagnosis,and the specificity was96.4%.In the PGL group,8(100%)patients with stage IIE PGL(n=8)and 6(46.1%)patients with stage IE PGL(n=13)were positive for monoclonal IgH gene rearrangements.CONCLUSION:IgH gene rearrangements may be associated with PGL staging and may be useful for the diagnosis of PGL and for differentiating between PGL and gastric linitis plastica.

Insertional mutagenesis and cloning of HIV env 125 peptide gene by polymerase chain reaction

One oligonueleotide probe,HIV env 101-1,and two oligonucleotide primers,HIV env125-1 and HIV env 125-2,were designed with the aid of a computer and synthesized by a DNAsynthesizer.The env 125 peptide gene was amplified by polymerase chain reaetion(PCR).Beingidentified by electrophoresis and Southern blotting,the PCR product was cloned into plasmidpUC 19.The recombinant pENV 125,identified with X-gal selection and restriction mapping, wassequenced.The results showed that the cloned env 125 peptide gene contained the inserted EcoRⅠ site and ATG at the 5’end,Hind Ⅲ site and TAG at the 3’end.The sequence and readingframe were proved to be correct.

Two－step multiplex polymerase chain reaction for gene diagnosis of progressive pseudohypertrophic muscular dystrophy

Ｔｗｏ－ｓｔｅｐｍｕｌｔｉｐｌｅｘｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎｆｏｒｇｅｎｅｄｉａｇｎｏｓｉｓｏｆｐｒｏｇｒｅｓｓｉｖｅｐｓｅｕｄｏｈｙｐｅｒｔｒｏｐｈｉｃｍｕｓｃｕｌａｒｄｙｓｔｒｏｐｈｙ￥ＴａｎＱｉｎｇｒｏｎｇ（谭庆荣）；ＷｕＢ...

Detection of Gene Alteration for Color Vision Defects by Polymerase Chain Reaction

According to the fact that the abnormalities of visual pigment genes were always involved in the changing of the exon 5, two oligonucleotide primers were designed to amplify the exon 5 of red pigment gene and green pigment gene. After electrophoresis of the PCR products digested with Rsal or Sau3A, the DNA fragments from the exon 5 of red pigment gene (RPG) and green pigment gene (GPG) were separated since there are different restriction endonuclease sites. On the other hand, we analyzed the exon 5 rela...

Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription

Quantitative real-time reverse transcription-polymerase chain reaction （qPCR） is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

STUDY ON CELL ORIGIN OF MALIGNANT LYMPHOPROLIFERATIVE DISORDERS WITH POLYMERASE CHAIN REACTION

Genotype of IgH, TCRγ and TCR δ gene rearrangement in 42 cases of malignant lymphoproliferative disorders were studied by using polymerase chain reaction (PCR) technique. The results suggested that among the 23 cases, in which malignant cells expressed B-lineage cell surface markers, 20 showed IgH gene rearrangement and 11 had TCRγ gene rearrangement and / or TCRδ gene deletion. All the 11 cases expressed T-lineage cell differentiation antigens were found to have TCRγand TCRδ gene rearrangement or deletion and only one had IgH gene rearrangement. Double rearrangements of IgH and TCRγ genes were detected in all the 3 cases of T and B double-phenotype ALL. In the cases malignant cells did not express any lineage specific antigens while 4/5 had TCRγ gene rearrangement but all failed in IgH gene rearrangement. The relation of cellular differentiation origin and rearrangement of antigen receptor genes with clinical manifestations was discussed.

Detection and amplification of Helicobacter pylori urease gene A in gastric biopsies by using nested polymerase (?)hain reaction

A nested polymerase chain reaction(N-PCR)for the spegific detection of Helicobacterpylori(H.pylori)was developed with two primer pairs(nested primers)derived from ureasegene A of H.pylori.The N-PCR was used to detect 21 different samples of H.pylori including20 clinical isolates and 1 reference strain NCTC 14126,but it was negative for other bacterialspecies,showing the N-PCR assay to be 100% specific.Tenfold serial dilution experiments re-vealed the detection of as little as 0.1 fg of H.pylori DNA by N-PCR.To evaluate the PCR as-say for clinical samples,gastric biopsies were tested with N-PCR,and the results were comparedwith those of culture,urease test and histologic examination(reference standard,RS).In 30biopsy specimens,H.pylori DNA sequences were detected by PCR in all of 20(100%)positivetissue and none of the 10 negative tissues.PCR is a specific and sensitive method that can detectthe presence of H.pylori without the need for culture and would have significant importance di-agnostically and epidemiologically.

Detection of immunoglobulin gene rearrangement in leukemia by seminested PCR amplification and Southern blot hybridization

ＤｅｔｅｃｔｉｏｎｏｆｉｍｍｕｎｏｇｌｏｂｕｌｉｎｇｅｎｅｒｅａｒｒａｎｇｅｍｅｎｔｉｎｌｅｕｋｅｍｉａｂｙｓｅｍｉｎｅｓｔｅｄＰＣＲａｍｐｌｉｆｉｃａｔｉｏｎａｎｄＳｏｕｔｈｅｒｎｂｌｏｔｈｙｂｒｉｄｉｚａｔｉｏｎＸｕＢｉｎｇ（徐兵）；ＺｈｏｕＳｈ...

Cloning of polyadenylated mRNA fragments of Escherichia coli with restriction display-polymerase chain reaction

Objective:To investigate the polyadenylation of mRNA in E. Coli. Methods: The mRNA of E. Coli was enriched from the total RNA with oligo(dT)-cellulose, prior to reverse transcription using oligo(dT)18as the primer. Double-stranded cDNA was subsequently synthesized, which was subjected to digestion with Sau3A I to produce multiple gene fragments for ligation with the adapters. PCR was carried out in 10 groups according to 10 different pairs of the selective primers, and the PCR products were then cloned into T-vectors. Results: More than 100 gene fragments had been cloned, 30 of which were sequenced. Conclusion:Polyadenylation of E. Coli mRNA may not be a biochemical curiosity but a general attribute of bacterial mRNA.

Gene Rearrangement Analysis of Orbital Lymphoid Infiltrating Disorders

Purpose:To determine whether the use of polymerase chain reaction for B-cell gene rearrangement in patients with orbital lymphoid infiltrate disorders could be useful in the diagnosis of lymphoma,especially,in differentiating benign lesion from malignant one.Methods:In addition to clinical,pathological,and immunohistochemical evaluations,48 cases of orbital lymphoid infiltrate disorders were examined for immunoglobulin heavy(IgH)gene rearrangement by means of PCRto amplify the FR3region with formalin-fixed and paraffin-embedded tissues.Results:Gene rearrangement in the third frame-work of the IgH region was detected in specimens obtained from15cases of malignant lymphoma,4of reactive lymphoid hyperplasia and3of orbital pseudotumor.All of these patients showed a discrete band(100bp)which reflected monoclonal proliferation of B lymphocytes.5cases of malignant lymphoma,6ofreactive lymphoid hyperplasia and 15of orbital pseudotumor did not show a discrete band on PCR.Conclusions:The FR3 region gene rearrangement of Ig heavy in patients with orbital lymphoid infiltrate disorders may be an additional diagnostic tool in differentiating benign from malignant lymphoid diseases and in offering a useful adjunct for diagnosis in difficult or unclear cases.It is a reliable and practical method of gene diagnosis in orbital lymphoid infiltrate disorders and helps to identify the molecular mechanism of malignant lymhoma.Eye Science2000;16:15-21.

Bcl-2 GENE REARRANGEMENT DETERMINED BY PCR AS AMEAN TO DETECT MINIMAL RESIDUAL DISEASE INMALIGNANT LYMPHOMAS

Objective: To develop a sensitive method to detect minimal residual disease and to elucidate the significance of bcl-2 gene rearrangement in diagnosis and treatment of malignant lymphoma. Methods: Using polymerase chain reaction (PCR) to detect bcl-2 gene rearrangement and using serial dilution method to define the sensitivity of PCR. Results: In 9 different malignant lymphoma cell lines, Su-DHL-4 and Su-DHL-6 were shown bcl-2(MBR)/JH rearrangement, the sensitivity of PCR was 1:105. In 16 patients with follicular lymphoma, the peripheral blood and bone marrow were PCR positive in 4 cases both at initial diagnosis and after complete remission. Conclusion: Detection of bcl-2 gene rearrangement by PCR provides a sensitive and specific assay of minimal residual disease. It is helpful to improve staging of disease, prognosis and evaluation of the treatment results.

Detection of IgH and T-cell receptor gene rearrangement in leukemia by PCR amplification and its clinical application

PCR amplification was used to detect immunoglobulin heavy chain (IgH), T-cell receptor (TCR)Vγ1-Jγ, Vδ2-Dδ 3 rearranged genes in 45 cases of acute lymphoblastic leukemia (ALL) and IgH gene rearrangement in 41 cases acute nonlymphoblastic leukemia (ANLL). The positive percentage of the IgH, TCR Vγ1-Jγ and Vδ 2-Dδ3 gene rearrangement was found to be 66. 7%, 62.2%, and 35. 6%, respectively in 45 ALL patients. Multiple rearranged IgH gene bands were found in 8 (26. 7% ) out of 30 ALL patients with IgH gene rearrangement. IgH gene rearrangemens were found in 7 (17. 1% ) out of 41 ANLL patients. ALL patients with positive IgH rearranged gene were with B cell and those with positive TCR Vγ1-Jγ gene rearrangements and with negative IgH rearranged gene were with T-ALL,those with positive both IgH and TCR Vδ2-Dδ3 rearranged genes mostly were with preB-ALL. The complete remission (CR) rate was low in ALL patients with multiple rearranged heavy chains and ANLL patients with IgH gene rearrangement, who were more likely to have a clinical relapse, which may be due to clonal evolution. In complete remission (CR) patients, the cytologic relapse fell behind postive PCR results for an average periods of 7. 2 weeks (range 2 to 23 weeks). Using PCR to detect rearranged genes could be helpful in determining the efficacy of chemotherapy, predicting clinical relapse, assisting gene typing and predicting prognosis.

Analysis of gene rearrangements of antigen receptors of nonHodgkin＇s lymphoma of T－cell lineage

ＡｎａｌｙｓｉｓｏｆｇｅｎｅｒｅａｒｒａｎｇｅｍｅｎｔｓｏｆａｎｔｉｇｅｎｒｅｃｅｐｔｏｒｓｏｆｎｏｎＨｏｄｇｋｉｎ＇ｓｌｙｍｐｈｏｍａｏｆＴ－ｃｅｌｌｌｉｎｅａｇｅ￥ＺｈａｎｇＪｉａｎｚｈｏｎｇ（张建中）；ＹａｎＬｉａｎｇｓｕｉ（晏良遂）；ＢａｉＹ...

DETECTION OF BCL-2 GENE MAJOR BREAKPOINT REGION REARRANGEMENT IN HUMAN B-CELL LYMPHOMAS

Objective To investigate the frequency of t(14; 18) in different subtypes of B-cell lymphomas and the ability or the polymerase chain reaction(PCR) to detect this rearrangement in frozen samples. Methods 1o7 cases of B-cell lymphomas were studied uslng DNA extracted from rresh-frozen tissues. The DNA samples were amplified by PCR for bcl-2 MBR/JH. The products of bcl-2/JH rearrangement were hybridized with an internal olignucleotide probe or bcl-2 MBR. Results The rearranged bcl-2MBR/JH gene was detected in 13 of the 25(52. o% ) follicular center lymphomas, according to REAL classification: 8 of 11 (72. 7%) grade 1, 2 of 5(40. 0%) grade I, and 3 of 90 (33. 3%) grade, 17 of 82(2o. 8%) cases or difruse large B-cell lymphomas were found to have detectable bel-2 MBR/J. rearrangement- Conclusion The rrequency or bcl-2 MBR/JH rearrangement in diffuse large B-cell lymphomas is significantly lower than those in follicular center lympkomas(X2= 9. 28, P <o. oo5), suggesting that bcl2/JH rearrangements occur mainly in follicular center lymphomas. in addition, the result of reconstruction experiments suggest that amplification or bcl-2 MBR/JH rearrangements by PCR is both sensitive and specific for detection of t (14; 18 ) translocation.

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.

RT-qPCR normalization of reference genes in different lifehistory stages of Gracilaria vermiculophylla(Rhodophyta)

The quantitative realtime polymerase chain reaction(RT-qPCR)is a powerful and sensitive method to measure expression of targeted gene but it highly relies on the use of suitable reference genes for data normalization.We evaluated the expressions of 8 housekeeping genes:18S ribosomal rDNA(18S rDNA),28S ribosomal r DNA(28S rDNA),rubisco large subunit(rbc-L),β-actin(ACT),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),elongation factor 1(EF1),β-tubulin(Tub B),and P-phycoerythrin B(PEB),to select the suitable reference genes for different life-history stages(tetrasporophyte,carposporophyte,and male/female gametophyte)of Gracilaria vermiculophylla by absolute quantitative method.Softwares geNorm and BestKeeper were used to verify the results acquired from copy number analysis.Results show that the expression of identified reference genes varied in comparing groups composed of different type of life stages.It is suggested that 18S rDNA and TubB could be used for highly complex samples composed of mixed ploidy and phases.18S rDNA and 28S rDNA were also preferred for using among the matured isomorphic samples.But for samples with different maturities,TubB and ACT were recommended for tetrasporophytes and gametophytes respectively.

土壤固碳微生物的绝对定量检测实验设计

为了定量检测土壤中的固碳微生物,设计以功能基因cbbL为靶标的固碳微生物微滴数字聚合酶链式反应(ddPCR)检测方法。选择合适的引物探针,从退火温度、探针浓度以及引物浓度进行反应条件的优化,分析ddPCR检测方法的线性范围、敏感性、重复性和特异性。结果显示,当退火温度为55.8℃、探针与引物浓度分别为350、750 nmol/L时,建立的cbbL-ddPCR扩增反应效率最高,阴阳性微滴分布界限最明显,平均拷贝数较高;检测的线性范围为2.3×10^(0)~2.3×10^(5)copies/μL-DNA,曲线方程y=0.1077x-95.562,相关系数R^(2)为0.9997,检出限为0.5 copy/μL-DNA,21个重复的变异系数仅为3.92%,与其他4种非固碳微生物DNA未发生交叉反应。所建立的cbbL-ddPCR方法可用于土壤微生物固碳潜能测定。

仰口线虫PCR检测方法的建立及应用

为特异性检测羊感染仰口线虫的情况,基于仰口线虫ITS基因序列设计特异性引物,进行退火温度及反应条件的优化,建立其属特异性PCR检测方法。应用该方法对仰口线虫、食道口线虫、毛尾线虫、捻转血矛线虫、细颈线虫、奥斯特线虫、夏伯特线虫、筒线虫、毛圆线虫的DNA样本进行扩增,仅能特异性扩增出仰口线虫的目的条带;该方法成功从仰口线虫中扩增出约500 bp的特异性片段,最低检测浓度为3.6×10^(3)copies/μL;该PCR检测方法对临床奶山羊粪便样品的检测结果显示,羊粪便样品中仰口线虫卵的阳性率为10%,且与显微镜镜检所得的结果一致。结果表明,建立的仰口线虫PCR检测方法敏感性较高且特异性良好,可以应用于临床羊粪便样品中仰口线虫的虫卵检测,为羊仰口线虫病的诊断与防治提供了一种技术支持。