Bp Polymerase Chain Reaction Product Research Articles

Isolation and characterization of a carboxysome shell gene from Thiobacillus neapolitanus.

The gene coding for the major carboxysome shell peptide (csoS1) from Thiobacillus neapolitanus has been isolated and sequenced. Oligonucleotide primers for polymerase chain reaction (PCR) amplification of the 5' end of the gene were made possible by amino acid sequencing of the N-terminal residues of the shell peptide. A 41 bp PCR product was used as a probe to isolate the gene. The deduced amino acid composition of the 216 bp gene shows a high degree of hydrophobicity. The gene is located within a series of three repeated regions of DNA and appears to have arisen via gene duplication. The transcript of csoS1 is approximately 400 bases in length. The shell peptide shares significant homology with Synechococcus open reading frames implicated in carboxysome structure/assembly. These open reading frames and csoS1 are related and are probably members of a carboxysome gene family.

Detection of Epizootic Hemorrhagic Disease Virus Serotypes 1 and 2 in Cell Culture and Clinical Samples Using Polymerase Chain Reaction

The potential for the use of the polymerase chain reaction (PCR) in detecting epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell cultures and clinical samples was studied. Using oligoribonucleotide primers selected from genome segment 6 of EHDV-2 (Alberta strain), the PCR-based assay resulted in a 387-base pair (bp) PCR product. EHDV RNA from US prototype serotypes 1 and 2 and a number of EHDV field isolates, propagated in cell cultures, were detected by this EHDV PCR-based assay. Amplification products were visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the PCR assay was 100 fg of virus RNA (equivalent to 6 x 10(3) virus particles) with ethidium bromide-stained agarose gels. Chemiluminescent hybridization increased the sensitivity of the PCR assay 1,000 times, and specific signals were detected from 0.1 fg of virus RNA (equivalent to 6 virus particles). Amplification product was not detected when the PCR-based assay was applied to RNA from the US bluetongue (BT) virus prototype serotypes 2, 10, 11, 13, and 17 or total nucleic acid extracts from uninfected baby hamster kidney-21 cells, Vero cells, and blood cells from deer that were EHDV seronegative and virus isolation negative. Application of this EHDV PCR-based assay to clinical samples resulted in detection of EHDV RNA from blood and spleen samples from a deer in California with clinical hemorrhagic disease. This EHDV PCR-based assay could provide a rapid, sensitive, and specific assay for detection of EHDV infection in susceptible ruminants.

Enzymatic amplification of mini-exon-derived RNA gene spacers of Leishmania donovani: primers and probes for DNA diagnosis

The multicopy mini-exon-derived RNA (med RNA) locus of Leishmania donovani was enzymatically amplified by the polymerase chain reaction (PCR). The major 180 bp PCR product contained conserved med RNA gene sequences flanking the variable intergenic spacer from the med RNA gene tandem repeat. The oligonucleotide primers cross-reacted with other Leishmania species. In serial dilution experiments, positivity in the PCR assay was observed down to the genomic DNA equivalent of less than a single Leishmania cell. When the major PCR products from Indian L. donovani isolates were cloned and used as probes in dot hybridization analyses, they discriminated between L. donovani and L. amazonensis, L. major and L. infantum under high stringency conditions. DNA from spleen biopsies and blood samples of confirmed kala azar patients was positive, as were two skin biopsies from patients with post-kala azar dermal leishmaniasis (PKDL). These observations demonstrate that PCR amplification of med RNA intergenic spacers is sufficiently sensitive for clinical diagnosis of kala azar and PKDL, and furthermore, that cloned intergenic spacer probes may be useful for identification and classification of L. donovani.

Alternative splicing of the p53 tumor suppressor gene in the Molt-4 T-lymphoblastic leukemia cell line

The expression of the p53 tumor suppressor gene in ten human cell lines (nine cancers and one normal) was studied using reverse transcription, polymerase chain reaction (PCR) and direct sequencing. Using P53U and P53D primers for amplifying a 371-base pair (bp) target fragment spanning exons 7–10 of p53 cDNA, normal-sized PCR products were amplified from 9 cell lines but not from the Hep3B hepatocellular carcinoma (HCC) cell line. An additional larger band (504 bp) was observed for the Molt-4 T-lymphoblastic leukemia cell line. Employing P531 and P53D primers which flank a 76-bp p53 cDNA fragment, 76 bp as well as 209 bp products were generated by PCR of Molt-4 cDNA. Direct sequencing of the 504 bp and 209 bp bands confirmed the presence of a 133 bp insertion between exons 9 and 10 in the aberrant transcript. This insertion was homologous to a 130-bp sequence within the wild-type p53 intron 9, except for 2 point mutations and 3 base insertions. Sequencing of P53U P53D PCR products of Molt-4 genomic DNA revealed an 8 bp deletion just downstream to the 133 bp insertion, creating a novel donor splicing site within intron 9. This site, coupled with an inherent acceptor splicing site just upstream to the 133 bp insertion, suggests that the 133 bp stretch represents an alternative exon. The occurrence of a termination signal within this alternative transcript is predicted to culminate in a truncated p53 translational product. The sequences of the 371 bp PCR products of Molt-4, HT-1080, SiHa, CaSki, HeLa and MRC-5 cell lines corresponded with the wild-type p53 cDNA. G → T transversions at the third base of codon 249 of p53 were detected in Mahlavu and PLC/PRF/5 HCC lines, while a TAC to CAC mutation at codon 234 was observed in an allele of the Raji Burkitt lymphoma line.

Isolation by asymmetric polymerase chain reaction and partial sequencing of the common bean chloroplast trnL (UAA) gene and pseudogene

AbstractA polymerase chain reaction (PCR) strategy has been developed which utilizes the common bean (Phaseolus vulgaris) chloroplast DNA as a template and primers from (a) the 5′ exon of the broad bean chloroplast trnL (UAA) gene (EMBL ac no. X51471) and (b) the 3′ exon of the common bean chloroplast trnL (UAA) pseudogene (EMBL ac no. X58537). A 600 bp PCR Product was reamplified using the 5′ exon primer and an internal primer from the 5′ end of the intron of the common bean chloroplast trnL (UAA) pseudogene. A 130 bp PCR Product was separated into 130 bp and 106 bp DNA fragments on an 8% non‐denaturing polyacrylamide gel. Both fragments were purified by phenol extraction, reamplified by asymmetric PCR reactions and directly sequenced by the Sanger method. When compared to the broad bean trnL (UAA) gene, the 130 bp PCR product showed sequence homologies of 100% with its 5′ exon and 79% over the first 58 nucleotides of the 5′ end of the intron. The 106 bp PCR product contained deletions in its 5′ exon region. These results indicated that the PCR technique had isolated the trnL (UAA) gene and its pseudogene.

The presence of messenger RNA for HLA class I in human platelets and its capability for protein biosynthesis

In order to determine whether platelets contain specific messenger RNA encoding for HLA class I molecules, polymerase chain reaction (PCR) was performed with RNA from different platelet donors. Two amplified 300 bp and 279 bp cDNA fragments were obtained which encompassed sequences from 321 to 620 and from 795 to 1073. The 300 bp fragment encodes exon 2 and exon 3, the 279 bp encodes a portion of exon 4, exon 5, exon 6 and a portion of exon 7. A 300 bp nested PCR product from one donor, that encoded for the highly polymorphic region alpha 2, was cloned and sequenced. The resulting nucleotide sequences fitted to the expected sequence for HLA B*3801 of this donor. Sequence analysis of the 279 bp PCR product demonstrated the presence of exon 5 encoding for the 117 bp transmembrane domain. In addition, de novo protein biosynthesis was studied by radioimmunoprecipitation of HLA class I molecules from 35S-methionine metabolically labelled platelet lysates with a monoclonal antibody (mab) w6/32 specific for a monomorphic epitope on the heavy chain of HLA class I antigens. Analysis of the immunoprecipitates on SDS polyacrylamide gel electrophoresis showed a specific band with apparent molecular weight (M(r)) of 44 kD corresponding to integral membrane HLA protein. On the basis of these results, we conclude that platelets contain specific messenger RNA encoding for HLA class I molecules and have the capability to synthesize the integral HLA membrane protein.

Quantitation of IL-4 expression in small numbers of cells from mice

Interleukin-4 (IL-4) is an important T cell and mast cell product that participates in allergic and cytotoxic responses, as well as functions as a growth factor for B, T, and inflammatory cells. Studies of the expression of IL-4 by T cells present in inflammatory reactions would be facilitated by using polymerase chain reaction (PCR) coupled to reverse transcription of mRNA to amplify the small quantity of mRNA present in these cells. In order to use this method in a quantitative manner, a plasmid was constructed that contained a modified form of mouse IL-4 cDNA. This plasmid was transcribed to produce cRNA for this modified sequence. The cRNA was used as an internal standard for the reverse transcription and amplification of IL-4 transcripts in RNA samples from mouse thymocytes. Amplification of reverse-transcribed native IL-4 mRNA produced a 286 bp PCR product. Amplification of the reverse-transcribed standard RNA produced a 155 bp product, which reflected a deletion introduced into the original IL-4 cDNA sequence. Comparison of the amount of the 286 bp native product to the amount of 155 bp standard product enabled the quantitative determination of IL-4 expression in each sample. This method was used to demonstrate that platelet activating factor increases the expression of IL-4 in mouse thymocytes and in a mouse T cell line. The expression of IL-4 by thymocytes exposed to platelet-activating factor (PAF) may reveal an important link between inflammation and the maturation of T cells in the thymus.

Identification of a nonsense mutation at amino acid 584–Arginine of platelet glycoprotein IIb in patients with type I Glanzmann thrombasthenia

Using Southern blot, the restriction digests of genomic DNAs in 11 patients with Glanzmann thrombasthenia from 10 unrelated kindreds were probed with a full-length GPIIb cDNA. An additional 2.3 kb Taq I fragment and two 1.65 kb and 0.65 kb fragments with reduced band intensity were found in the genes of two affected siblings from a family originating from the city of Huang Yan in the Zhejiang province. The Taq I digest of the abnormal gene was further probed with three portions of GPIIb cDNA, revealing that the heterozygous mutation was present in the region around exons 15-17 of the GPIIb gene. Two primers for polymerase chain reaction (PCR) were then designed, and a 394 bp PCR product was generated and sequenced, indicating that a stop codon (TGA) was substituted for an Arg codon (CGA) at amino acid position 584 of GPIIb, and resulted in a premature termination of translation and production of a shortened protein. The Western blot analysis showed that GPIIIa at the platelet surface was apparently deficient, it may be ascribed to the rapid turn-over of GPIIIa uncomplexed with the truncated GPIIb. The abnormal 2.3 kb Taq I fragment was used as a specific genetic marker to detect the carrier status of the patient family. The abnormal allele was proved to be derived from the mother, the two affected siblings are double heterozygotes, and one clinically unaffected daughter has also inherited this defective allele, while the father carries another recessive abnormal allele unidentified.

Quantification of polymerase chain reaction products in agarose gels with a fluorescent europium chelate as label and time-resolved fluorescence spectroscopy.

We have 5'-end-labeled one polymerase chain reaction (PCR) primer with the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). After performing PCR in the presence of another unlabeled primer, we separated the 362 bp PCR product with 2% low melting point agarose gel electrophoresis. The gel was then immersed into a Eu3+ solution. During soaking, Eu3+ diffuses into the gel and associates with BCPDA to form a fluorescent complex of long fluorescence lifetime. This complex can be quantified by scanning the gel with a time-resolved fluorometric reader. Because BCPDA and Eu3+ are not fluorescent by themselves, background signals are very low. The detection limit was about 5 ng of DNA. We have also shown that the BCPDA-labeled product could be blotted and detected on the membrane by using an anti-BCPDA antibody. These two technologies may find applications other than in PCR, e.g. in fluorescence-based DNA sequencing and in solution hybridization.

Detection and differentiation of picornaviruses in clinical samples following genomic amplification

A polymerase chain reaction (PCR) assay was used to detect and differentiate picornaviruses (PVs), using primers homologous to the 5' non-coding and VP2 regions of the PV genome. The PCR resulted in a 530 bp PCR product for human rhinoviruses (HRVs) and a 650 bp product for polioviruses, coxsackieviruses (CV) or echoviruses. The PCR assay could detect as little as 1 p.f.u. of virus in either cerebrospinal fluid (CSF) or stool, using ethidium bromide-stained gels. Standard strains of poliovirus, CV, echovirus and HRV were detected, with the exception of echovirus type 22. In contrast, heterologous viruses, such as herpes simplex virus, human cytomegalovirus, adenovirus, influenza virus and rotavirus, as well as human and monkey cell DNA, were not amplified. In nasal swabs taken from patients with respiratory infections, the PCR detected 27 of 28 HRV isolation-positive specimens. All specimens from which viruses other than HRVs were isolated were negative by PCR. The PCR definitively identified poliovirus and CVs from the CSF or stool of patients with aseptic meningitis, as well as CV in the pericardial fluid of a patient who had suffered a myocardial infarction. Specimens taken from patients with similar pathologies, and from which heterologous viruses were isolated, were uniformly negative by PCR.