Specific Reverse Primers Research Articles

Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D. nodosus in sheep with footrot prior to vaccination

The identification of Dichelobacter nodosus present in a flock is a prerequisite to specific (autogenous) vaccination. Current methods of identification of the serogroup present in a population requires that the organisms be isolated, identified visually in mixed culture on streak plates, subcultured to purify and subjected to antigenic analysis. This process takes at least 3 to 4 weeks. This study describes the development of a simple and rapid serogroup specific PCR test forD. nodosus . A common forward primer was designed from the conserved amino-terminal region of the fimbrial gene (fimA) and 9 (A–I) serogroup specific reverse primers were designed from the carboxy-terminal regions offimA of the different serogroups. To verify the specificity within D. nodosus, each specific primer pair was tested in PCR against 18 serogroups/serotypes (prototypes) and found to be specific for all the serotypes within the homologous serogroups. Eighty four other bacterial strains, either commonly occurring in sheep or found in the environment of sheep, and including organisms related taxonomically to D. nodosus, were used to check the specificity of these assays. They were found to be specific for D. nodosus as none of the 84 bacterial stains reacted. These primers detected 1pg of purified chromosomal DNA, or 50–100 cells of D. nodosus in crude lysates. Sensitivity was markedly improved when an immuno-magnetic capture was employed. Single tube multiplex PCRs were tested with different combinations of common forward primer and groups of 3, 4 or 5 reverse primers chosen so that amplicon size for each reaction product was different. These were able to amplify DNA of isolates from all the relevant serogroups included in the reactions. These tests were evaluated with samples taken directly from lesions of footrot, either directly or preceded by DNA purification, immuno-magnetic capture, enrichment broth culture and culture on hoof agar media. Of these methods only PCR on mixed colonies from 4-day-old cultures on 4% hoof agar media yielded results of practical value.

Identification of a novel HLA-B*07 allele--HLA-B*0726.

We describe here, the identification of a novel HLA-B*07 allele named HLA-B*0726. This allele was found in a Caucasian individual serologically typed as HLA-B7, B35. Novel DNA probe patterns for the HLA-B*07 allele were found using HLA-B specific reverse sequence-specific oligonucleotide probe (SSOP) and sequence-specific primer (SSP) typing. DNA sequencing demonstrated the presence of a new HLA-B*07 sequence variant encoding a single nucleotide substitution from a G to a T at nucleotide 539 in exon 3. This results in an amino acid substitution from arginine to leucine at residue 156 in exon 3.

The Human Serotonin 5-HT4 Receptor Regulates Secretion of Non-amyloidogenic Precursor Protein

The serotonin 5-HT(4) receptor has recently gained a lot of attention for its functional roles in central processes such as memory and cognition. In this study, we show that activation of the human 5-HT(4) (h5-HT(4)) receptor stimulates the secretion of the non-amyloidogenic soluble form of the amyloid precursor protein (sAPPalpha). 5-HT enhanced the level of secreted sAPPalpha in a time- and dose-dependent manner in Chinese hamster ovary cells stably expressing the h5-HT(4(e)) receptor isoform. The increase was inhibited by the selective 5-HT(4) receptor antagonist, GR113808. The 5-HT(4) selective agonists, prucalopride and renzapride, also increased secreted sAPPalpha in IMR32 human neuroblastoma cells. The stimulatory effect of 5-HT was mimicked by forskolin, a direct activator of adenylyl cyclase, and 8-bromo-cAMP, a membrane-permeant cAMP analogue. On the contrary, inhibition of protein kinase A (PKA) by H89 potentiated the 5-HT-induced increase in both secreted and cellular sAPPalpha. This phenomenon involves a novel PKA-independent stimulatory process that overcomes a PKA-dependent inhibitory one. Finally, activation of the h5-HT(4(e)) receptor did not modify extracellular amyloid beta-protein in Chinese hamster ovary cells transfected with the human APP695. Given the neuroprotective and enhancing memory effects of sAPPalpha, our results may open a new avenue for the treatment of Alzheimer's disease.

Preimplantation genetic diagnosis (PGD) of myotonic dystrophy (DM) by triplet primed PCR (TP-PCR): a protocol applicable to all at-risk couples.

Objective: Positive selection of unaffected embryos in the course of DM PGD has so far been based on the detection of the normal allele of the transmitting parent. This indirect diagnosis has mostly been carried out using the morbid CAG locus. From our sample of patients, about 1/4 of the couples were fully informative or non informative and 1/2 were semi informative. For the latter, half of the healthy embryos cannot be diagnosed, leaving for transfer only 25% of the starting embryos. This sort of diagnosis is therefore applicable satisfactorily to only 1/4 of the at risk couples. Our objective was to develop a simple and reliable single cell assay which can be applicable to all DM couples. Design: We wanted to develop a test allowing the detection of the pathological allele in DM. DM alleles can expand up to several thousands triplets, and, at the moment, the largest alleles can not always be PCR amplified even from milligrams of DNA. We therefore decided to amplify only part of the expansion using an oligonucleotide primer internal to the repeat itself as described in Warner et al. (1996). We needed to modify their protocol to achieve reliable amplification from single DNA copies. [∗Warner JP, Barron LH, Goudie D, Kelly K, Dow D, Fitzpatrick DR, Brock DJ. (1996) J Med Genet. 33 (12):1022–6.] Materials/Methods: Single cells were lysed in alkaline buffer (200 mM KOH, 50 mM DTT) and amplified with the expand Long template Taq polymerase and buffer II (Roche diagnosis). Hemi-nested amplification was carried out in a 9700 PCR system with a specific forward primer (O/I F) and the reverse triplet specific reverse primer elongated in 3′ with 20 nucleotides constituting a reverse inner primer. The inner amplification contained the O/I F primer fluorescently labeled with 6 FAM and the tail part only of the triplet primer. Twenty and twenty five thermal cycles were carried out for the outer and the inner reactions respectively. Fluorescent TP-PCR products were analyzed in a ABI 310 DNA analyzer. Each run takes about 30 minutes, so, for semi informative patients we decided to speed up the process by analyzing the PCR products by non fluorescent polyacrylamide gel electrophoresis and only running the non informative allelic combinations in the ABI 310. Results: Expanded alleles were detected in 100% of the lymphocytes analysed and one PGD cycle was carried out for a semi informative couple. Four embryos were biopsied and only two yielded a PCR product from each of their 2 biopsied blastomeres. In all 4 cells, only the shared allele was detectable after traditional gel electrophoresis. After fluorescent analysis there was no trace of TP amplification in any of the cells and both embryos were transferred. Positive hCG were measured in the first dosage but had decreased in the second. Ten blastomeres were desaggregated from the two remaining embryos and all positive cells showed TP-amplified product indicating the presence of the expanded allele in both these embryos. Conclusions: Reliable detection of expanded CAG repeats was achieved from single lymphocytes and blastomeres using fluorescent TP-PCR. One chemical pregnancy was achieved using this diagnosis technique. Supported by: This work was supported in part by the association Française contre les myopaties (AFM) and l’assistance publique des hopitaux de Paris (AP-HP).

Transcription Factor BACH1 Is Recruited to the Nucleus by Its Novel Alternative Spliced Isoform

The transcription factor Bach1 is a member of a novel family of broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) basic region leucine zipper factors. Bach1 forms a heterodimer with MafK, a member of the small Maf protein family (MafF, MafG, and MafK), which recognizes the NF-E2/Maf recognition element, a cis-regulatory motif containing a 12-O-tetradecanoylphorbol-13-acetate-responsive element. Here we describe the gene structure of human BACH1, including a newly identified promoter and an alternatively RNA-spliced truncated form of BACH1, designated BACH1t, abundantly transcribed in human testis. The alternate splicing originated from the usage of a novel exon located 5.6 kilobase pairs downstream of the exon encoding the leucine zipper domain, and produced a protein that contained the conserved BTB/POZ, Cap'n collar, and basic region domains, but lacked the leucine zipper domain essential for NF-E2/Maf recognition element binding. Subcellular localization studies using green fluorescent protein as a reporter showed that full-length BACH1 localized to the cytoplasm, whereas BACH1t accumulated in the nucleus. Interestingly, coexpression of BACH1 and BACH1t demonstrated interaction between the molecules and the induction of nuclear import of BACH1. These results suggested that BACH1t recruits BACH1 to the nucleus through BTB domain-mediated interaction.

Expression of Vascular Endothelial Growth Factor mRNA in GI-101A and HL-60 Cell Lines

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that has a strong association with growth and metastasis of various cancers. We analyzed the expression of VEGF mRNA levels in human breast-tumor derived GI-101A cells and in human promyelocytic leukemia derived HL-60 cells using RT-PCR technique. During our RT-PCR analysis we detected the expression of three splice variants of VEGF mRNA at 400, 520 and, 650 bp lengths, which were amplified by a single set of VEGF specific forward and reverse primers. The three RT-PCR products detected by us in these cells correspond to the mRNA splice variants coding for the three isoforms of VEGF respectively, VEGF121, VEGF165, and VEGF189. Treatment of GI-101A and HL-60 cells with phorbol 12, 13-dibutyrate (PDB) or diethylstilbestrol (DES) resulted in a significant increase of VEGF mRNA levels in a dose dependent manner. Both treatments increased the levels of all three splice variants of VEGF mRNA and a maximum increase was detected with 10 μM concentrations of PDB or DES treatments after 2 h. Interestingly, both PDB and DES mediated stimulation of VEGF mRNA expression was completely blocked by the PKC inhibitor chelerythrine. Quantitation of VEGF levels by ELISA technique confirmed that changes seen in mRNA levels following different treatments altered the release of VEGF. Our results suggest that PDB and DES mediated effects on VEGF expression in GI-101A and HL-60 cells occur at the gene transcription level.

Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains.

Taking advantage of point mutations between DNA sequences of closely related microbial strains, PCR primers modified with respect to the target sequence at positions 2-5 near the 3' end were designed to obtain a fragment harbouring an artificial restriction site specific for a given strain. The modified forward primer coupled with a specific reverse primer allows for the amplification of DNA fragments which can be digested with the specific endonuclease only in those strains where the restriction site is inserted by the DNA polymerase. The effectiveness of the method, named restriction site insertion-PCR (RSI-PCR), was tested on isolates of the 'Bacillus cereus group' for the rapid typing and discrimination of these closely related strains.

Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains

Taking advantage of point mutations between DNA sequences of closely related microbial strains, PCR primers modified with respect to the target sequence at positions 2–5 near the 3′ end were designed to obtain a fragment harbouring an artificial restriction site specific for a given strain. The modified forward primer coupled with a specific reverse primer allows for the amplification of DNA fragments which can be digested with the specific endonuclease only in those strains where the restriction site is inserted by the DNA polymerase. The effectiveness of the method, named restriction site insertion-PCR (RSI-PCR), was tested on isolates of the ‘ Bacillus cereus group’ for the rapid typing and discrimination of these closely related strains.

Detection of COL1A1-PDGFB fusion transcripts in dermatofibrosarcoma protuberans by reverse transcription-polymerase chain reaction using archival formalin-fixed, paraffin-embedded tissues.

The reciprocal translocation t(17;22)(q22;q13) and a supernumerary ring chromosome, r(17;22), derived from the translocation, have been shown to be highly characteristic of dermatofibrosarcoma protuberans (DFSP). Its consequence is a fusion of two genes, a collagen type I alpha 1 gene (COL1A1) and platelet-derived growth factor B-chain gene (PDGFB). The COL1A1-PDGFB fusion gene, is expected to be a diagnostic molecular assay. However, previous studies on this subject were mostly based on frozen tissue specimens or cultured tumor cells. In this present study, the investigators conducted a reverse transcription (RT)-polymerase chain reaction (PCR) assay to detect the COL1A1-PDGFB fusion transcripts using archival formalin-fixed, paraffin-embedded tumor specimens from 12 patients with DFSP. To amplify the fusion transcripts, a specific COL1A1 forward and PDGFB reverse primers were designed for single step PCR. The COL1A1-PDGFB fusion transcripts could be detected in 10 of 12 paraffin-embedded DFSP tumor specimens (83%). Subsequent sequence analysis using the PCR products confirmed that the detected messages were derived from gene fusions composed of PDGFB exon 2 and different regions of the COL1A1 gene (exon 8, 10, 22, 24, 32, 38, 45 or 46). Two samples of Bednar tumor included in this series also contained the fusion transcripts. In sample of DFSP with fibrosarcomatous transformation, the COL1A1-PDGFB could not be detected in the fibrosarcoma areas of the third recurrence, though the chimeric transcripts were identified in the ordinary DFSP areas of the first recurrence. No fusion transcripts could be amplified in non-DFSP lesions, including 10 dermatofibromas and 9 malignant fibrous histiocytomas. These results indicate that this molecular assay could be applied to archival formalin-fixed, paraffin-embedded tumor tissues as a diagnostic aid for DFSP.

Detection of unculturable bacteria in periodontal health and disease by PCR.

Recently developed molecular methods have made it possible to characterize mixed microflora in their entirety, including the substantial numbers of bacteria which do not grow on artificial culture media. In a previous study, molecular analysis of the microflora associated with acute oral infections resulted in the identification of three phylotypes, PUS3.42, PUS9.170, and PUS9.180, representing as-yet-uncultured organisms. The aim of this study was to design and validate specific PCR primers for these phylotypes and to determine their incidences in samples collected from healthy and diseased periodontal tissues. Two specific reverse primers were devised for each phylotype, and these were used in duplex PCRs with universal forward and reverse primers. All three phylotypes were detected in periodontal sites; PUS9.170, related to oral asaccharolytic Eubacterium spp., was significantly associated with disease. This study demonstrates the possibility of using unculturable, and therefore uncharacterized, organisms as markers of disease.

Engineering and expression of a full length cDNA encoding Schistosoma japonicum paramyosin : Purification of the recombinant protein and its recognition by infected patient sera

A cDNA encoding the complete open reading frame of the Schistosoma japonicum paramyosin has been constructed and cloned. The 2600 bp cDNA was engineered by PCR using a N-terminally truncated paramyosin clone (pmy25) as template and a 57-mer primer that introduced the eight missing amino acids and matched the 5′ sequence of pmy25 in conjunction with a pmy specific reverse primer. After cloning and expression, the recombinant protein was purified by affinity chromatography under non-denaturing conditions and was shown to have a molecular mass of 99 kDa which is equivalent to the expected size of the full length recombinant fusion protein, comprising the 97 kDa paramyosin plus an additional 2 kDa for the N-terminal fusion peptide incorporating the six histidine residues required for purification. In Western blot assays it reacted specifically with anti-paramyosin antibodies in sera from vaccinated animals and patients with Asian schistosomiasis. The engineering of the full-length cDNA encoding Schistosoma japonicum paramyosin, its bacterial expression and purification will facilitate future studies aimed at determining its efficacy as an anti-schistosomiasis vaccine.

A tyrosine kinase profile of prostate carcinoma.

Tyrosine kinases play central roles in the growth and differentiation of normal and tumor cells. In this study, we have analyzed the general tyrosine kinase expression profile of a prostate carcinoma (PCA) xenograft, CWR22. We describe here an improved reverse transcriptase-PCR approach that permits identification of nearly 40 different kinases in a single screening; several of these kinases are newly cloned kinases and some are novel. According to this, there are 11 receptor kinases, 9 nonreceptor kinases, and at least 7 dual kinases expressed in the xenograft tissue. The receptor kinases include erbB2, erbB3, Ret, platelet-derived growth factor receptor, sky, nyk, eph, htk, sek (eph), ddr, and tkt. The nonreceptor kinases are lck, yes, abl, arg, JakI, tyk2, and etk/bmx. Most of the dual kinases are in the mitogen-activating protein (MAP) kinase-kinase (MKK) family, which includes MKK3, MKK4, MEK5, and a novel one. As a complementary approach, we also analyzed by specific reverse transcriptase-PCR primers the expression profile of erbB/epidermal growth factor receptor family receptors in a variety of PCA specimens, cell lines, and benign prostatic hyperplasia. We found that erbB1, -2, and -3 are often coexpressed in prostate tissues, but not in erbB4. The information established here should provide a base line to study the possible growth and oncogenic signals of PCA.

Expression of Choline Acetyltransferase mRNA and Protein in T-Lymphocytes.

Acetylcholine (ACh) is present in human blood and localized in T-lymphocytes which exhibit ACh-synthesizing activity. As the final step towards clarifying the origin of ACh present in the blood, we elucidated the enzyme involved in ACh synthesis in T-lymphocytes by detecting the mRNA for choline acetyltransferase (ChAT), which is known to catalyze ACh synthesis in the nervous system. The ChAT mRNA in the MOLT-3 human leukemic T-cell line was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using specific order and reverse primers. A single specific RT-PCR product was observed on agarose gel. The sequence of the RT-PCR product of MOLT-3 was completely identical with nucleotide positions 322-973 of human brain ChAT cDNA. Western blot analysis with an antibody to ChAT confirmed the presence of ChAT protein in MOLT-3. These findings demonstrate that mRNA for the same ChAT as that in the nervous system is expressed in T-lymphocytes, and indicate that ACh synthesized by ChAT in T-lymphocytes is the origin of ACh in the blood.