Polymerase Chain Reaction Oligonucleotide Research Articles

Circulating Blood B Cells in Multiple Myeloma: Analysis and Relationship to Circulating Clonal Cells and Clinical Parameters in a Cohort of Patients Entered on the Eastern Cooperative Oncology Group Phase III E9486 Clinical Trial

Recent analyses of circulating blood B cells in myeloma have generated controversy concerning the exact levels of these cells and whether they may represent circulating clonal tumor B cells. Previous reports suggested that CD19+ B cells are markedly increased in myeloma patients and that this population shares clonotypic rearrangements with the malignant plasma cell. We studied the numbers of CD19+ B cells by flow cytometry in previously untreated newly diagnosed myeloma patients in Eastern Cooperative Oncology Group (ECOG) phase III trial E9486. There were 628 patients who were eligible for the clinical protocol E9486, but of these 521 were also entered on the companion laboratory study (E9487) and had CD19 data. In comparison with normal controls, the myeloma patients exhibited a marked heterogeneity in the number of circulating CD19+ B cells as detected by flow cytometry. Approximately 20% of patients had significantly increased levels of circulating CD19+ B cells. However, the total CD19+ blood population from myeloma was not significantly different from the median of age-matched, normal controls. Analysis of CD19+ blood cells in relationship to circulating clonal cells was done in 13 myeloma patients using a clonotypic, quantitative allele-specific oligonucleotide-polymerase chain reaction (PCR) assay. No correlation was found between the numbers of CD19+ B cells (range, 5% to 51%) and PCR estimates of the number of clonal cells in the peripheral blood (range, .009% to 3.6%). Low CD19+ B-cell level (<125 μL) was associated with clinical stage III (P = .033). A significant relationship exists between higher levels (≥125/μL) of CD19 cells and longer overall survival (P < .0001). In addition, high CD19 levels also predicted a clinical response and longer event-free survival. There was a strong inverse association between the level of CD19 values at diagnosis and infections within the first 2 months of diagnosis. Importantly, the number of deaths related to infections was significantly greater in the low versus high CD19 group (P < .0202). Also, CD19 is an independent prognostic factor in addition to plasma cell labeling indices, β2 -microglobulin, hemoglobin, and plasmablastic morphology. Patients with infections were more likely to have low levels of CD19+ cells. In summary, higher CD19+ cell levels are a favorable prognostic sign with no apparent relationship to circulating tumor cells. In addition, this analysis strongly suggests that low peripheral blood levels of CD19+ cells are an adverse prognostic sign in myeloma. The CD19+ cell levels in myeloma patients is an important parameter in the overall assessment of these patients.

Detection of mutations in PCR products from clinical samples by surface plasmon resonance.

Two different strategies for scanning and screening of mutations in polymerase chain reaction (PCR) products by hybridization analysis are described, employing real-time biospecific interaction analysis (BIA) for detection. Real-time BIA was used to detect differences in hybridization responses between PCR products and different 17-mer oligonucleotide probes. For the analysis using a biosensor instrument, two different experimental formats were investigated based on immobilization of either biotinylated PCR products or oligonucleotide probes onto a sensor chip. Applied on the human tumour suppressor p53 gene, differences in hybridization levels for full-match and mismatch situations employing both formats allowed the detection of point mutations in exon 6 PCR products, derived from a breast tumour biopsy sample. In addition, a mutant sample sequence could be detected in a 50/50 background of wild type exon 6 sequence. The suitability of the different formats for obtaining a regenerable system and a high throughput of samples is discussed.

Detection of hepatitis B virus transcriptional activity: An hypothesis

This paper outlines a method of demonstrating hepatitis B virus (HBV)-related transcriptional activity based on the random amplification of cDNA ends (RACE) protocol. Following RNA extraction from the appropriate tissue, cDNA is synthesized using a long oligo d(T) based primer containing unique 5' oligonucleotide sequences. Nested polymerase chain reaction (PCR) is then carried out using virus-specific oligonucleotides and standard PCR (∼20 base pairs) oligonucleotides homologous to 5' regions of the long oligo-d(T) primer used to initiate the cDNA reaction. This technique is particularly applicable to the study of HBV transcription in small samples of tissue, for example, from renal biopsies obtained from patients with HBV-related nephritis.

Detection of Clostridium botulinum type F using the polymerase chain reaction

The polymerase chain reaction (PCR) was used to amplify a portion of the Clostridium botulinum type F toxin gene. An 1137-bp fragment was amplified from 11 different strains of type F C. botulinum with primers derived from the published sequence of type F strain no. 202. This fragment was not amplified from the DNA of C. botulinum types A, B and E, or from other clostridial organisms examined. When used as a hybridization probe, the 1137-bp PCR-generated fragment generated from one of the type F strains (the proteolytic strain type F Langeland) hybridized to the PCR products from all other type F toxin-producing strains tested. Portions of fragments amplified from the type F Langeland strain were sequenced. The sequence of this strain was found to exhibit approximately 3% variation from the published sequence of the non-proteolytic type F strain no. 202. Primers designed to pair with the regions of maximum sequence variation between strain 202 and the Langeland strain gave amplification products only with DNA from type F strains that exhibited the same proteolytic properties as the strain from which the primer sequences were derived. These findings underscore the need to consider variations in sequence when designing oligonucleotide probes and PCR primers in order to avoid false negative results.

P53 mutation, expression, and DNA ploidy in evolving gliomas: evidence for two pathways of progression.

Two lines of evidence indirectly implicate the tumor suppressor p53 (also known as TP53) gene in glioma development. First, germline mutations of the p53 gene are associated with increased susceptibility to glioma. Second, chromosome 17p deletions and p53 gene mutations are found frequently in sporadic gliomas of all malignancy stages. These observations suggest that mutations of the p53 gene may be early events in glioma development. Our purpose was to analyze 15 low-grade astrocytic gliomas that progressed to higher-grade gliomas, examining the status of the p53 gene in both the initial and recurrent tumors. Also, we explored the relationships between p53 status, DNA ploidy, tumor grade, and patient survival. Fifteen low-grade gliomas that recurred as tumors of higher grade 17-102 months after initial treatment (biopsy, resection, radiotherapy, or chemotherapy) were identified from hospital records of patients (eight male and seven female) aged 31-68 years. Pathologic diagnosis was re-evaluated. Polymerase chain reaction (PCR)-single-strand conformation polymorphism and DNA sequencing were performed on tissue samples from the initial and recurrent tumors of each patient, using oligonucleotide PCR primers directed to exons 5-9 of the p53 gene. p53 expression was determined by immunohistochemistry and DNA ploidy evaluated by DNA flow cytometry. Eight (53%) of fifteen tumors had p53 mutations in exons 5-9. Nine (64%) of fourteen were immunopositive initially, and eight of these were also immunopositive at recurrence. p53 gene status was significantly associated with p53 expression in the initial tumor (P = .02), and p53 expression at initial diagnosis was significantly related to tumor pathology at recurrence (P = .03). Patients with p53 mutant tumors survived nearly twice as long as those without mutations (median survival, 61 versus 33 months; P = .031). There was no significant difference in recurrence-free survival between patients with p53 mutant and nonmutant tumors (48 versus 33 months; P = .37), but there was a significant difference in postrecurrence survival (17 versus 2 months; P = .019). Low-grade tumors that recurred as anaplastic gliomas were characterized by p53 gene mutation, immunopositivity, and DNA non-diploidy. Low-grade tumors that recurred as glioblastomas generally had intact p53 genes and were immunonegative. These findings suggest that histologically indistinguishable, low-grade astrocytic gliomas that are destined to progress to higher grades, do so along two distinct clinicopathologic pathways (either stepwise to anaplastic glioma, then glioblastoma, or directly to glioblastoma) marked by the presence or absence of p53 mutation.

Design and characterization of PCR primers for detection of pathogenic Neisseriae

Oligonucleotide polymerase chain reaction (PCR) primers directed at a group of closely related Neisserial species were designed from 165 rDNA sequences even though only a single such sequence from the targeted group was known. The amplifiable group included all Neisserial species considered pathogenic for man, including Neisseria gonorrhoea and Neisseria meningitidis. None of 43 other bacterial DNA specimens were amplified, including five non-Neisserial Neisseraciae and three non-pathogenic Neisseriae. Another non-pathogenic Neisserial species gave a signal only at high DNA concentrations. DNA specimens from the pathogenic Neisseriae were detectable in amounts as low as 0·001 pg per PCR reaction, the approximate equivalent of a single organism, with equal sensitivity in buffer or in simple extracts of human inflammatory synovial fluids to which Neisserial DNA had been added. Simultaneously studied control specimens lacking added DNA were negative. The approach used to design these group-directed primers using only a single rDNA sequence from the targeted group by exploiting known patterns of sequence conservation among the 16S rDNA genes may prove useful for designing other similar group-directed primers. Polymerase chain reaction primers prepared in this way should prove of value in a number of areas, both investigational and clinical.

Identification of Enteropathogenic Campylobacter Species by Oligonucleotide Probes and Polymerase Chain Reaction Based on 16S rRNA Genes

Summary On the basis of 16S ribosomal RNA (rRNA) sequences published for Campylobacter species C. jejuni, C. colt, C. fetus and C. hyointestinalis , we were able to design three oligonucleotide probes (probes 6-1, 10-1 and 18-1r) specific only for C. jejuni and C. coli . 16S rRNA genes of 60 different Campylobacter strains were amplified by the Polymerase Chain Reaction (PCR) using universally conserved primers and the amplification products were hybridized with the probes. This analysis showed that probe 6-1 hybridizes with the 16S rRNA from C. jejuni, C. coli, C. lari and C. upsaliensis ; probe 10-1 hybridizes with 16S rRNA from C. jejuni, C. coli and C. lari , while probe 18-1r hybridizes with 16S rRNA from C. jejuni, C. coli, C. lari and some strains of C. upsaliensis . When the oligonucleotides 6-1 and 18-1r are used as primers in PCR amplification, and the resulting PCR product is hybridized with the internal probe 10-1, the DNA equivalent of two bacteria can be detected specifically for the group of pathogenic species C. jejuni, C. coli , and C. lari .

Feline cytokines TNFα AND IL‐1β: PCR cloning and sequencing of cDNA

Abstract Using oligonucleotide primers and polymerase chain reaction (PCR), cDNAs for feline cytokines TNFα and IL‐1β were amplified, cloned, and sequenced. The cDNA for PCR amplification was prepared from mRNA derived from lipopolysaccharide (LPS) stimulated feline bone marrow derived macrophages. PCR was performed using sets of oligonucleotide primers designed to specifically amplify cDNAs for IL‐1β or TNFα. PCR fragments were cloned into pGEM 3ZF(‐) or pCR 1000 vectors, sequenced and consensus nucleotide sequences reported. The cDNA for feline TNFα had a 98.6% match with coding regions of a genomic clone for feline TNFα which was recently reported (McGraw, 1990). The two feline TNFα clones differ by 8 nucleotide base pair (bp) changes which result in 5 amino acid differences in the predicted protein sequence. A search of GenBank and EMBL determined that the feline TNFα cDNA consensus sequence had a 90, 86, 85, 82 and 83 percent overall match with human, porcine, ovine, mouse and goat TNFα cDNAs, respec...

Genotyping of the aldehyde dehydrogenase 2 (ALDH2) gene using the polymerase chain reaction: Evidence for single point mutation in the ALDH2 gene of ALDH2-deficiency

About half of all Japanese lack the activity of aldehyde dehydrogenase 2 (ALDH2), and suffer a flush after alcohol intake due to the marked elevation of blood acetaldehyde concentration. The cause of ALDH2 deficiency is thought to be a single point mutation in codon 487 of the ALDH2 gene. However, this mutant ALDH2 gene has not yet been cloned and sequenced. We amplified and cloned the exon 12 of the ALDH2 gene using polymerase chain reaction (PCR), and revealed that normal GAA coding glutamic acid is replaced for AAA coding lysine in codon 487 of the mutant ALDH2 gene. Based on this finding, we performed the genotyping of the ALDH2 gene using PCR and allele-specific oligonucleotide probes. The genotypes of 13 subjects with ALDH2-active phenotype were all homozygous for the normal ALDH2 gene (ALDH2(1)), while in 9 subjects with ALDH2-deficient phenotype 2 subjects were homozygous for the mutant ALDH2 gene (ALDH2(2)) and the other 7 subjects were heterozygous for both genes, indicating that the mutant ALDH2 gene is dominant. In 20 normal control subjects, the prevalence of ALDH2(1)/ALDH2(1), ALDH2(1)/ALDH2(2) and ALDH2(2)/ALDH2(2) was 45%, 45% and 10% respectively. On the other hand, in 36 alcoholic liver disease patients, the prevalence of the genotypes was 83%, 17% and 0%. These results confirmed the previous observation that the incidence of ALDH2 deficiency is much lower in alcoholic liver disease patients than in the general population, and suggested that most of the ALDH2 deficient patients with alcoholic liver disease are heterozygous for the normal and mutant ALDH2 genes.

Olga—oligonucleotide primer design program for the Atari ST

A program to facilitate the design of oligonucleotide primers has been devised. Olga is written in draft ANSI standard 'C' and makes use of the implementation of Digital Research GEM (Graphics Environment Manager) on the Atari ST. Olga is specifically suited to the polymerase chain reaction (PCR) allowing simultaneous analysis of two primer sequences. The advantage of Olga is that it provides in one program analyses for direct repeats, secondary structures and primer dimerization as well as several useful 'finishing' tools for workers engaged in PCR optimization and oligonucleotide syntheses.

A Novel Method for Detecting Point Mutations or Polymorphisms and Its Application to Population Screening for Carriers of Phenylketonuria

We describe a method termed PCR (polymerase chain reaction) amplification of specific alleles (PASA), a generally applicable technique for detection of point mutations or polymorphisms. The ease and technical simplicity of PASA will make genetic analyses more accessible to the general medical community. In addition, PASA shows promise for population screening because the technique is rapid, highly reproducible, inexpensive, nonisotopic, and amenable to automation. PASA is a modification of PCR that depends on the synthesis of a PCR oligonucleotide primer that precisely matches with one of the alleles but mismatches with the other. When the mismatch occurs near the 3' end of the PCR primer, amplification is inefficient. Therefore, preferential amplification of the perfectly matched allele is obtained. We demonstrate the applicability of PASA by performing carrier detection in the family of a patient with phenylketonuria (PKU) and by screening a population of unrelated subjects for the presence of the two mutations most commonly associated with PKU. Multiple persons were screened simultaneously for the mutant alleles because a mutation could be detected in the presence of at least a 40-fold excess of the normal allele. The two PKU mutations could be detected concurrently by using a mixture of only three PCR primers, an indication that simultaneous screening of multiple mutations can be done even if three or more mutations are closely clustered. In addition to the detection of mutations, PASA can be used to detect polymorphic alleles rapidly and to distinguish pseudogenes or repetitive sequences that differ by as little as one base.