Endoglin inhibits prostate cancer motility via activation of the ALK2-Smad1 pathway.

Endoglin is a transforming growth factor beta (TGFbeta) superfamily auxiliary receptor. We had previously shown that it suppressed prostate cancer (PCa) cell motility, and that its expression was lost during PCa progression. The mechanism by which endoglin inhibits PCa cell motility is unknown. Here we demonstrate that endoglin abrogates TGFbeta-mediated cell motility, but does not alter cell surface binding of TGFbeta. By measuring Smad-specific phosphorylation and Smad-responsive promoter activity, endoglin was shown to constitutively activate Smad1, with little-to-no effect upon Smad3. Knockdown of Smad1 increased motility and abrogated endoglin's effects. As type I activin receptor-like kinases (ALKs) are necessary for Smad activation, we went on to show that knockdown of ALK2, but not TGFbetaRI (ALK5), abrogated endoglin-mediated decreases in cell motility and constitutively active ALK2 was sufficient to restore a low-motility phenotype in endoglin deficient cells. These findings provide the first evidence that endoglin decreases PCa cell motility through activation of the ALK2-Smad1 pathway.

Temporal changes in renal endoglin and TGF-beta1 expression following ureteral obstruction in rats.

Chronic renal disease is characterized by the accumulation of extracellular matrix proteins in the kidney and a loss of renal function. Tubulointerstitial fibrosis has been reported to play an important role in the progression of chronic renal diseases. Transforming growth factor-beta1 (TGF-beta1) is a profibrotic cytokine playing a major contribution to fibrotic kidney disease. Endoglin is a membrane glycoprotein of the TGF-beta1 receptor system. The aim of this work was to determine the time-course expression of renal type I and IV collagens, endoglin and TGF-beta1 in a rat model of induced tubulointerstitial fibrosis at 1, 3, 10 and 17 days after unilateral ureteral obstruction (UUO). In 17 days-ligated (L)-renal samples, a marked interstitial fibrosis was detected by Masson's trichromic and Sirius red staining, accompanied by an increase in type I collagen expression as shown by immunohistochemical analysis. Northern blot studies revealed a progressive increase in collagen alpha2(I), TGF-beta1 and endoglin mRNA expression in L kidneys when compared with the corresponding non-ligated (NL) kidneys from the animals subjected to left UUO. Seventeen days after UUO, significant increases in collagen alpha2(I), collagen alpha1(IV), TGF-beta1 and endoglin mRNA levels were detected in L kidneys vs NL kidneys. Significantly higher levels of the protein endoglin were found in L kidneys than in NL kidneys 10 and 17 days following obstruction. A marked increase expression for endoglin and TGF-beta1 was localized in renal interstitium by immunohistochemical studies 17 days after obstruction. In conclusion, this work reports the upregulation of endoglin coincident to that of its ligand TGF-beta1 in the kidneys of rats with progressive tubulointerstitial fibrosis induced by UUO.

Up-regulation of endoglin, a TGF-beta-binding protein, in rats with experimental renal fibrosis induced by renal mass reduction.

BACKGROUND: The central process in chronic renal failure is the progressive accumulation of extracellular matrix in the glomeruli and in the tubulo-interstitial space, resulting in renal fibrosis. Transforming growth factor-beta1 (TGF-beta1) up-regulation plays a major role in the genesis of renal fibrosis. Endoglin is a membrane glycoprotein that binds TGF-beta1 and TGF-beta3 with high affinity. An increased level of endoglin immunostaining has been demonstrated previously in biopsies from patients with chronic progressive renal disease. We have assessed the expression of endoglin in the rat 5/6th renal mass reduction (RMR) model. METHODS: One, 3 and 5 months after RMR, mean arterial pressure and renal function were measured, animals were sacrificed, renal fibrosis was evaluated quantitatively and the expression of endoglin was assessed by western blot, northern blot and immunohistochemistry. RESULTS: RMR induced a progressive increase in mean arterial pressure and urinary protein excretion. Renal corpuscular area, and mesangial and interstitial fibrosis increased with time after RMR. Immunohistochemical staining for endoglin demonstrated its expression mainly on the endothelial surface of major vessels. In kidneys 1 and 3 months after RMR, the expression of endoglin in renal corpuscles was limited to Bowman's parietal epithelium. In rats 5 months after RMR, the immunoexpression in glomerular endothelium was more marked. Northern blot analysis revealed that rats with RMR showed an increase in the expression of mRNA for endoglin, only at 5 months after RMR. Western blot analysis gave a different time course: a marked increase in the first month, a decrease in the 3rd month and a further increase in the 5th month after RMR. CONCLUSIONS: The present study demonstrates increased endoglin expression in rats with severe hypertension and renal damage. This increased endoglin expression coincides with the period of higher renal damage and renal dysfunction.

Coordinate expression of novel genes during osteoblast differentiation.

To achieve new insights into the coordinate regulation of gene expression during osteoblast differentiation we utilized an approach involving global analysis of gene expression to obtain the identities of messenger RNAs (mRNAs) expressed using an established in vitro model of bone development. MC3T3-E1 osteoblast-like cells were induced to differentiate by the addition of beta-glycerophosphate (beta-GP) and ascorbic acid. RNA samples derived from induced and uninduced control MC3T3-E1 cells were used to prepare complementary DNA (cDNA) for serial analysis of gene expression (SAGE). A preliminary SAGE database was produced and used to prepare a hybridization array to further facilitate the characterization of changes in the expression levels of 92 of the SAGE-mRNA assignments after induction of osteoblast differentiation, specifically after 6 days and 14 days of ascorbate treatment. SAGE-array hybridization analysis revealed coordinate induction of a number of mRNAs including Rab24, calponin, and calcyclin. Levels of MSY-1, SH3P2, fibronectin, alpha-collagen, procollagen, and LAMPI mRNAs, present at day 6 postinduction, were markedly reduced by day 14 postinduction. A number of unanticipated and potentially important developmental genes were identified including the transforming growth factor beta (TGF-beta) superfamily member Lefty-1. Lefty-1 transcript and translation product were found to be induced during the course of MC3T3-E1 cell differentiation. We present evidence, using transient transfection and antibody neutralization approaches, that Lefty-1 modulates the induction of alkaline phosphatase (ALP) after treatment of MC3T3-E1 cells with ascorbate and beta-GP. These data should provide useful new information for future analysis of transcriptional events in osteoblast differentiation and mineralization.

Chemiluminescent determination of hydrogen peroxide with 9-acridinecarbonylimidazole and use in measurement of glucose oxidase and alkaline phosphatase activity.

Several activated derivatives of 9-acridinecarboxylic acid were prepared in order to investigate their utility for detection of hydrogen peroxide. One of these derivatives, 9-acridinecarbonylimidazole (I), is especially stable and is a useful reagent for measuring, by chemiluminescence, the activity of a number of enzymes that directly produce peroxide, including glucose oxidase. Other enzymes can also be assayed if an appropriate intermediate substrate exists that ultimately produces hydrogen peroxide after being acted upon by the enzyme. 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and 3-indolyl phosphate (3-IP) are such substrates for alkaline phosphatase. The detection limits for both of these enzymes are in the 1-10 amol range. Other enzymes that can potentially be assayed using I include oxidases, hydrolases and dehydrogenases. Negative assays for compounds that consume or bind peroxide such as reducing agents, antioxidants, catalases and peroxidases are also feasible.

Involvement of Ets transcription factors and targets in osteoblast differentiation and matrix mineralization.

The osteoblast-like MC3T3-E1 cell line provides an excellent in vitro model of bone development. This system undergoes three orderly time-dependent phases characterized by proliferating preosteoblasts, matrix accumulation by postmitotic differentiating osteoblasts, and mineralization of the matrix, which results in the formation of multilayered bone nodules. The Ets family transcription factors regulate genetic programs that affect the proliferation and differentiation of osteoblasts. Of the eight Ets family transcription factors examined by our laboratory, only Etsl and Ets2 were found to be expressed at significant levels in this osteogenic system. Etsl is expressed in proliferating preosteoblastic cells, whereas Ets2, silent during this phase, is expressed by differentiating and mature osteoblasts. In addition, the expression of Etsl can be induced in MC3T3-E1 and fetal rat calvaria cells by retinoic acid (RA) which is known to exert profound effects on skeletal growth and development and bone turnover and induce specific cellular responses in bone cells. Thus, the multiple functions of RA in bone cells are likely to be mediated in part by Etsl. We show that the expression of Ets2 precedes and then parallels osteopontin expression and that the OPN promoter contains Ets binding sites and is a transcriptional target of Ets2. In order to identify other potential Ets target genes, we analyzed promoter regions of genes revealed by serial analysis of gene expression as present in the differentiation stage. The functional analysis of these genes has the potential to provide much needed information as to their function in osteogenesis and mineralization of the extracellular matrix and in bone-related diseases.

HLA-DMA allele polymorphism: no impact on kidney allograft outcome.

The purpose of this study was to analyze the association of HLA-DMA alleles with rejection episodes and early graft loss (EGL) in renal transplant recipients. One hundred and eighty four HLA-DMA alleles were retrospectively analyzed by DNA sequence analysis in 92 kidney transplant recipients. The gene frequencies of HLA-DMA *0101, *0102, *0103 and *0104 were found to be similar in all recipients, regardless of rejection vs non rejection episodes and EGL incidence. In conclusion, HLA-DMA allele polymorphism did not impact renal allograft outcome.

Endoglin, a TGF-beta receptor-associated protein, is expressed by smooth muscle cells in human atherosclerotic plaques.

Endoglin is a transmembrane protein that is found in association with transforming growth factor-beta (TGF-beta) superfamily receptor complexes and has an expression pattern that appears to be restricted primarily to endothelial cells, activated macrophages, trophoblasts, and fibroblasts. Since mutations in endoglin have been shown to be linked to hereditary hemorrhagic telangiectasia type 1, a disease manifested as vascular malformations characterized by excessive layers of vascular smooth muscle cells (VSMC), the expression of endoglin was investigated in VSMC. In vivo, the majority of SMC in human atherosclerotic plaques expressed high levels of endoglin, while endoglin was not detected in SMC from samples of the normal arterial wall. In vitro studies demonstrate that human aortic smooth muscle cells (HASMC) express the L-isoform of endoglin. Like endothelial cells, HASMC express endoglin protein as a dimer on the cell surface that binds TGF-beta1. In vitro, endoglin expression by HASMC is upregulated in response to TGF-beta1, suggesting that the presence of this factor in the atherosclerotic plaque might be responsible for the increased expression of endoglin. The demonstration of increased levels of endoglin in VSMC in human atherosclerotic plaques suggests a role for SMC endoglin in the maintenance of vascular integrity and in the response of the vessel wall to injury.

The potential role for prolactin-inducible protein (PIP) as a marker of human breast cancer micrometastasis.

The prolactin-inducible protein (PIP/GCPD15) is believed to originate from a limited set of tissues, including breast and salivary glands, and has been applied as a clinical marker for the diagnosis of metastatic tumours of unknown origin. We have investigated the potential role of PIP mRNA as a marker of human breast cancer metastasis. Using reverse transcription polymerase chain reaction and Southern or dot blot analysis, PIP mRNA was detected in 4/6 breast cell lines, independent of oestrogen receptor (ER) status. In breast primary tumours (n = 97), analysed from histologically characterized sections, PIP mRNA was detected in most cases. Higher PIP mRNA levels correlated with ER+ (P = 0.0004), progesterone receptor positive (PR+) (P = 0.0167), low-grade (P = 0.0195) tumours, and also PIP protein levels assessed by immunohistochemistry (n = 19, P = 0.0319). PIP mRNA expression was also detectable in 11/16 (69%) of axillary node metastases. PIP mRNA expression, however, was also detected in normal breast duct epithelium, skin, salivary gland and peripheral blood leucocyte samples from normal individuals. We conclude that PIP mRNA is frequently expressed in both primary human breast tumours and nodal metastases. However, the presence of PIP expression in skin creates a potential source of contamination in venepuncture samples that should be considered in its application as a marker for breast tumour micrometastases.

Soluble transforming growth factor-beta type II receptor inhibits negative remodeling, fibroblast transdifferentiation, and intimal lesion formation but not endothelial growth.

Using the rat balloon catheter denudation model, we examined the role of transforming growth factor-beta (TGF-beta) isoforms in vascular repair processes. By en face in situ hybridization, proliferating and quiescent smooth muscle cells in denuded vessels expressed high levels of mRNA for TGF-beta1, TGF-beta2, TGF-beta3, and lower levels of TGF-beta receptor II (TGF-betaRII) mRNA. Compared with normal endothelium, TGF-beta1 and TGF-beta2, as well as TGF-betaRII, mRNA were upregulated in endothelium at the wound edge. Injected recombinant soluble TGF-betaRII (TGF-betaR:Fc) localized preferentially to the adventitia and developing neointima in the injured carotid artery, causing a reduction in intimal lesion formation (up to 65%) and an increase in lumen area (up to 88%). The gain in lumen area was largely due to inhibition of negative remodeling, which coincided with reduced adventitial fibrosis and collagen deposition. Four days after injury, TGF-betaR:Fc treatment almost completely inhibited the induction of smooth muscle alpha-actin expression in adventitial cells. In the vessel wall, TGF-betaR:Fc caused a marked reduction in mRNA levels for collagens type I and III. TGF-betaR:Fc had no effect on endothelial proliferation as determined by reendothelialization of the denuded rat aorta. Together, these findings identify the TGF-beta isoforms as major factors mediating adventitial fibrosis and negative remodeling after vascular injury, a major cause of restenosis after angioplasty.

Cloning of the promoter region of human endoglin, the target gene for hereditary hemorrhagic telangiectasia type 1.

Endoglin (CD105) is a cell surface component of the transforming growth factor-beta (TGF-beta) receptor complex highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for the hereditary hemorrhagic telangiectasia type 1 (HHT1), also known as Osler-Weber-Rendu syndrome (OMIM 187300). This is an autosomal dominant vascular disorder probably caused by a haploinsufficiency mechanism displaying low levels of the normal protein. To understand the mechanisms underlying the regulated expression of endoglin, a genomic DNA clone containing 3.3 kb of the 5'-flanking sequence of the human endoglin gene has been isolated. The 5'-flanking region of the endoglin gene lacks consensus TATA and CAAT boxes, but contains two GC-rich regions and consensus motifs for Sp1, ets, GATA, AP-2, NFkappaB, and Mad, as well as TGF-beta-, glucocorticoid-, vitamin D-, and estrogen-responsive elements. As determined by primer extension and 5' RACE experiments, a cluster of transcriptional start sites was found to be located 350 bp upstream from the translation initiation codon. To analyze the endoglin promoter activity, the upstream -400/+341 fragment was fused to the luciferase gene and transient transfections were conducted in several cell types. This construct displayed a tissue-specific activity in human and bovine endothelial cells. Analysis of various deletion constructs showed the existence of a basal promoter region within the -81/+350 fragment as well as major transcriptional regulatory elements within the -400/-141 fragment. Electrophoretic mobility shift assays demonstrated the specific interaction of a member of the ets family with a consensus motif located at position -68. A promoter construct mutated at this ets sequence showed a much reduced activity as compared with the wild-type construct, supporting the involvement of this ets motif in the basal activity of the promoter. The endoglin promoter exhibited inducibility in the presence of TGF-beta1, suggesting possible therapeutic treatments in HHT1 patients, in which the expression level of the normal endoglin allele might not reach the threshold required for its function. Isolation and characterization of the human endoglin promoter represents an initial step in elucidating the controlled expression of the endoglin gene.

Mutation and expression analysis of the endoglin gene in hereditary hemorrhagic telangiectasia reveals null alleles.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and recurrent hemorrhage from the sites of vascular lesions. Two genes have been identified for HHT. Endoglin, a TGF-beta binding protein which maps to chromosome 9q3, is the gene for HHT1. The type and location of most of the previously described mutations in the endoglin (ENG) gene suggested a dominant-negative model of receptor-complex dysfunction for the molecular basis of this disorder. In this article we describe 11 novel ENG mutations in HHT kindreds, which include missense and splice-site mutations. Two identical missense mutations in unrelated families disrupt the start codon of the gene. In addition, some frameshift and nonsense mutations lead to very low or undetectable levels of transcript from the mutant allele. These combined data suggest that the nature of most ENG mutations is to create a null (nonfunctional) allele, and that there is no requirement for the synthesis of a truncated endoglin protein in the pathogenesis of HHT.

Allele-specific hybridization of lipoprotein lipase and factor-V Leiden missense mutations with direct label alkaline phosphatase-conjugated oligonucleotide probes.

Direct label alkaline phosphatase (AP) conjugated oligonucleotide probes (AP-DNA) were prepared to assess their utility for allele-specific detection of single base substitutions. Oligonucleotide conjugates were designed to detect point mutations in the genes for lipoprotein lipase (LPL) and coagulation factor-V (FV). Genomic DNA samples, including ones known to harbor point mutations in the genes for LPL and FV, were prepared from whole blood and subjected to polymerase chain reaction (PCR). PCR products were analyzed by Southern hybridization with the allele-specific AP-DNA probes and restriction endonuclease analysis. Thermal profiles for hybridization indicate optimal allele-specific selectivity was achieved with temperatures ranging from 45 degrees C to 55 degrees C at a total Na divided by concentration of 150 mM. Under these conditions the base changes studied were easily discriminated with allele specific hybridization signals in excess of 200:1 as estimated by scanning densitometry. Complete concordance was observed between hybridization and restriction analyses for 175 LPL and 201 FV clinical and reference samples. The total time for analysis of the PCR products was less than 2 h with a dot blot hybridization protocol.

Use of the polymerase chain reaction to specifically amplify integrated HPV-16 DNA by virtue of its linkage to interspersed repetitive DNA.

A polymerase chain reaction (PCR) based technique that combines a virus specific primer and a human interspersed repetitive sequence (IRS) specific primer in order to detect integration of human papilloma virus type 16 (HPV-16) is described. Amplification of viral-host DNA junctions occurs when viral integration results in placement of the virus specific primer binding site near (less that 3-4 kb) the primer binding site within a repetitive sequence element. The method relies on enzyme labeled oligonucleotide probes to achieve rapid, specific, and nonradioisotopic detection of viral integration related PCR products since episomal forms of the viral DNA do not lead to exponential accumulation of hybridizable PCR products. The technique is demonstrated for human genomic DNA derived from clinical cervical swab specimens and archival paraffin embedded blocks. Viral integration was detected in 41% of the HPV-16 positive samples (n = 34). In this positive subset, 64% were classified as invasive neoplasias, 29% CIN III and 7% CIN II. Analyzing the positive invasive neoplasias, 6 of 9 (66%) of the fingerprint results were obtained when an HPV primer was paired with an Alu primer. Interestingly, 100% of Alu primed fingerprint results obtained were derived from samples presenting invasive neoplasia (P < 0.025 by chi square).

Non-radioisotopic detection of human xenogeneic DNA in a mouse transplantation model.

Xenogeneic mouse models are widely used for the study of human tumor growth and metastasis. To date, few methods have been developed to track and quantitate the colonization of mouse organs with transplanted human cells. In this paper, a family of nonradioisotopic DNA oligonucleotide probes that are complementary to sequences within the human Alu element are characterized. These probes can be used in Southern hybridization reactions to quantitate the colonization of mouse organs with human derived cells. One oligonucleotide probe, the Alu-C probe, was identified as the most sensitive and specific in the family of probes synthesized for the distinction of human genomic DNA in a mouse genomic DNA background. The Alu-C probe can identify 0.05 ng human diploid DNA in a mouse background of 500 ng of genomic DNA. This represents 7.5 human diploid cells admixed with 75,000 mouse diploid cells. The Alu-C probe can therefore be employed to assess human colonization in xenograft models for a variety of human tumors and non-neoplastic tissues.

Hyperlipidemia and pancreatitis during pregnancy in two sisters with a mutation in the lipoprotein lipase gene.

OBJECTIVE: To explore genetic mechanisms for pregnancy-associated pancreatitis and hyperlipidemia in two sisters. DESIGN: Case history. SETTING: Tertiary care facility with outpatient follow-up. PATIENTS: Two sisters with acute pancreatitis and the acute respiratory distress syndrome were admitted (patient 1) or transferred (patient 2) to an intensive care setting with severely elevated triglyceride levels. Patient 1 was in the last trimester of pregnancy; patient 2 was 1 month postpartum. Both patients were of French Canadian ancestry. INTERVENTION: Acute treatment was directed at stabilizing both patients medically (with fat restriction) and one patient surgically (patient 2). Treatment with fat restriction, weight loss, and gemfibrozil was continued after hospitalization. RESULTS: Through DNA sequencing, we detected a mutation at amino acid residue 188 of lipoprotein lipase (LPL), reflecting product from one allele of the LPL gene in which a glutamine residue was substituted for a glycine (gly 188-->glu). CONCLUSION: LPL plays a key role in regulating triglyceride levels in pregnancy. Mutations of LPL may place the patient at risk for pancreatitis. This heterozygous LPL mutation, gly 188-->glu, is prevalent in certain ethnic groups and may be a common cause of pancreatitis associated with pregnancy.

An empirical method for the estimation of nonspecific PCR primer derived genomic reactivity.

A total of 36 PCR primers were subjected to thermal cycling, in a reaction composed of a single primer only, in order to assess their individual tendencies toward "nonspecific" amplification of human genomic DNA background. Nonspecific amplification of genomic DNA was estimated by Southern hybridization of the amplification products with an Alu-specific DNA probe. The yield of amplified Alu-hybridizing material was estimated by scanning densitometry. In this way a quantitative estimate of "mispriming" was obtained for the primers tested. Human Alu and total primate GenBank databases were scanned in order to obtain an estimate of the prevalence of homologous primer binding sites for each primer. Database searches were conducted at both 70% primer binding site homology and 100% homology at the 3'-terminal third of the primer. The observed levels of amplified, Alu-hybridizing material correlated best, but only marginally, with homology-based estimates of potential cross-reactivity at the 3' terminus of the primer (R = 0.193). A set of p53 tumor suppressor gene primers, exhibiting low and high extremes of background amplification, were tested for sensitivity in the presence and absence of added genomic DNA. The primer that gave the highest level of background amplification was the one whose performance was most severely affected by added genomic DNA. Empirical assessment of the tendencies of individual primers to amplify irrelevant DNA at low levels of the intended target may permit a useful "noise-specific" adjunct to primer design by computational methods.

Inhibition of DNA hybridization following partial dUTP substitution.

Carry-over contamination from PCR products can yield persistent false-positive results leading to significant problems in the resolution of true positive results. Commercial kits used in many laboratories help prevent carry-over contamination. We present data on the effects of dUTP substitution on the hybridization properties of amplified DNA using alkaline phosphatase conjugated oligonucleotide probes. We observe a pronounced depression in hybridization signal intensity in some dUTP-substituted PCR products. The magnitude of the decreased hybridization signal intensity appears proportional to both the dUTP concentration in the amplification reaction and the fraction of thymidylate residues in the probe binding site. The hybridization signal is nearly eliminated from PCR products synthesized in the presence of dUTP only and where the probe binding site is particularly rich in thymidylate residues. The decrease in hybridization signal is not always restored with less stringent hybridization conditions. Conditions that permit efficient hybridization and detection of bound probe but do not compromise carry-over protection are discussed.

The significance of platelets with increased RNA content (reticulated platelets). A measure of the rate of thrombopoiesis.

Direct flow cytometric measurement of nucleic acid content in individual platelets is possible using the fluorescent dye Thiazole Orange (Becton-Dickinson, San Jose, CA). When applied to studies of thrombocytopenic patients, platelets with elevated nucleic acid content ("reticulated platelets") can be identified and quantitated. Labeling of these platelets is saturable and is abolished by treatment with RNAse. It has been suggested that, similar to the erythrocyte reticulocyte response to anemia, the number of these platelets appearing in the circulation may provide an estimate of the rate of thrombopoiesis. The authors studied 229 thrombocytopenic patients, measuring both reticulated platelets and platelet-associated immunoglobulin. The results show that for the subset of patients with normal levels of platelet-associated immunoglobulin, the average absolute number of reticulated platelets is independent of platelet count and remains in the normal range. For those with elevated levels of platelet-associated immunoglobulin, the absolute number of reticulated platelets increases in patients who are moderately thrombocytopenic (60 to 100 x 10(9)/L) but decreases to normal or subnormal levels as thrombocytopenia worsens. The latter finding has been duplicated in studies of mice made thrombocytopenic by injection of anti-platelet antiserum. These results are consistent with the hypothesis that reticulated platelets are subject to peripheral destruction at the same rate as mature platelets, and that in the severely thrombocytopenic patient their level may decrease despite an appropriate marrow thrombopoietic response.

Triple-helical capture assay for quantification of polymerase chain reaction products.

This method for rapid, automated analysis of polymerase chain reaction (PCR) products makes use of PCR primers containing 5'-polypyrimidine sequences. Polypyrimidine-"headed" primers confer to the PCR product the ability to form triple helical complexes with a third polypyrimidine oligonucleotide. Third-strand oligonucleotides are modified to serve as either capture reagents or detection reagents for PCR products. Automated quantitative measurement of the PCR product is achieved by using latex bead-based fluorescence analysis. The use of triple-instead of double-helical interactions avoids the usual requirements of complex blocking reagents, time- and labor-intensive washing steps, and long times for color development. The method also provides rapid, sequence-specific capture and detection of PCR products without the need to denature the double-stranded PCR product. The assay is demonstrated with use of both PCR primer-derived and endogenous triple-helix-forming sequences resulting from PCR of several bacterial and viral target nucleic acids.