Detection of beta-thalassemia mutations by ASO hybridization of PCR amplified DNA with digoxigenin ddUTP labeled oligonucleotides.

A simple procedure for nonradioactive labeling of oligonucleotides has recently been developed (1). It consists of 3' end labeling of oligonucleotides with terminal transferase by incorporation of a single digoxigenin labeled dideoxy uridine triphosphate. We used these oligonucleotides for allele specific oligomer hybridization of polymerase chain reaction amplified DNA, followed by an enzyme-linked immunoassay and subsequent enzyme-catalyzed color reaction. We compared this procedure with the standard radioactive oligonucleotide hybridization technique through the detection of the most common Mediterranean beta-thalassemia mutations. This procedure was also used for the confirmation of a new mutation at position -87 (C----A) (2) of the beta-globin gene and for the subsequent family analysis.     

Hemoglobin Pasadena: identification of the gene mutant by DNA analysis using synthetic DNA probes

Hemoglobin Pasadena [beta 75(E19)Leu----Arg] was found in a boy who had an acute episode of anemia and rapid splenic enlargement. His father was the only other member of a large family with this hemoglobinopathy. We have used gene mapping techniques for direct identification of the beta-globin gene mutation. To correlate the DNA findings with the structural identification of this variant, we have also performed globin chain separation and analysis of the tryptic peptides using high performance liquid chromatography and secondary ion mass spectral analysis.     

Mutational analysis of beta-thalassemia cases from the Aegean region of Turkey using an allele-specific oligonucleotide hybridization technique

The aim of the present study was to evaluate the point mutations of beta-thalassemia patients from the Aegean region of Turkey by using an allele-specific oligonucleotide hybridization technique. DNA isolated from peripheral blood samples of 75 children with beta-thalassemia major or intermedia was analyzed using a Bio-Rad mD(x)(TM)-Be Tha Gene 1 kit. We determined mutations in 56 (74.6%) patients. The allelic frequency of mutations in 150 chromosomes was as follows: IVS-I-110 (G-A) 44.1%, IVS-I-1 (G-A) 28.2%, IVS-I-6 (T-C) 13.3%, IVS-II-745 (C-G) 9.3%, IVS-II-1 (G-A) 2.7%, Cd 39 (C-T) 2.4%, -87 (C-G) 0% and Cd 6 (-A) 0%. The distribution of the mutation types was consistent with the findings of other research groups.     

Hemoglobin Koln: direct analysis of the gene mutation by synthetic DNA probes

The molecular defect leading to Hb Koln has been analyzed by synthetic oligonucleotides. Thus, DNA of 19 nucleotides, in length corresponding to the normal and mutant beta-globin gene sequences, were used to develop a direct assay for the beta k-gene that codes for this most common form of the unstable hemoglobins. The use of synthetic oligonucleotides established that the Hb Koln mutation is due to a G-to- A transition. The conditions described here should result in the determination of all Hb Koln genotypes with a high level of confidence.     

Familial hypocalciuric hypercalcemia. Mild expression of the gene in heterozygotes and severe expression in homozygotes

Autosomal dominant familial hypocalciuric hypercalcemia was found in a kindred with neonatal severe primary hyperparathyroidism, previously judged to be an autosomal recessive trait. Mild hypercalcemia was documented in eight members representing three generations. Mild hypercalcemia was documented at an age as early as one week. In seven adults presumed to be heterozygotes, urinary calcium levels were in the same range as for familial hypocalciuric hypercalcemia. An additional adult member (who previously underwent parathyroidectomy for neonatal severe primary hyperparathyroidism) showed an abnormality in renal clearance of calcium and sodium characteristic of combined familial hypocalciuric hypercalcemia and surgical hypoparathyroidism. Parathyroidectomy in three hypercalcemic members did not cause normocalcemia. Unlike other kindreds with familial hypocalciuric hypercalcemia in whom hypercalcemia is consistent over time and moderate in heterozygotes, this kindred was characterized by heterozygotes showing hypercalcemia that was intermittent and mild. The consanguineous parents of the two previously described severely affected neonates were judged to be heterozygotes for familial hypocalciuric hypercalcemia. In conclusion, (1) a gene presenting as familial hypocalciuric hypercalcemia can be expressed as hypercalcemia that is intermittent and very mild in heterozygotes; (2) such a gene can cause neonatal severe primary hyperparathyroidism in homozygotes.     

A beta-thalassemia gene caused by a 290-base pair deletion: analysis by direct sequencing of enzymatically amplified DNA

The base composition around a recently detected deletion in the human beta-globin gene was determined by direct DNA sequencing of an enzymatically amplified DNA segment. The deletion removes 290 base pairs (bp), including the entire exon 1 and the mRNA cap site. In the vicinity of the deletion endpoints, the normal beta-globin gene contains direct and inverted repeats which may have taken part in generation of this deletion.     

Histologic typing of non-Hodgkin's lymphomas by in situ hybridization with DNA probes of oncogenes

Expression of six proto-oncogenes (fos, myc, myb, Ki-ras, Ha-ras, and N- ras) in 43 cases of non-Hodgkin's lymphoma was analyzed by means of in situ hybridization. Biotinylated DNA probes of the six oncogenes and those of immunoglobulin H (IgH) gene and T-cell receptor beta (TCR beta) chain gene were used. The results of in situ hybridization performed under blind conditions by IgH gene and TCR beta chain gene probes were compatible with those of typing by cell surface markers. The nuclear protein-related proto-oncogenes, fos, myc, and myb, were expressed in about 70% to 80% of all cases regardless of phenotype, histology, or histologic grade. On the contrary, genes of ras family were expressed in more limited numbers of cases except for the Ki-ras gene, which was more frequently expressed by cases of the T-cell immunophenotype with a high malignancy grade. The results of dot hybridization with RNA extracted from some cases were compatible with those of in situ hybridization, further demonstrating the specificity of in situ hybridization.     

Reinterpretation of G-banded complex karyotypes by fluorescence in situ hybridization with chromosome-specific DNA painting probes and alpha-satellite centromere-specific DNA probes in malignant hematological disorders

In this study, we have performed fluorescence in situ hybridization (FISH) with chromosome-specific DNA painting probes 1, 2, 3, 4, 6, 8, and 12 and centromere-specific DNA probes 7, 10, 12, 17, 18, and X after G-banding on the same metaphase spreads from four patients with malignant hematological disorders to more precisely interpret their complex karyotypes. The findings demonstrated that the application of combined G-banding and FISH can more accurately explain complex karyotypes of hematological malignancies. FISH can detect not only the origin of marker chromosomes, but also the complex rearrangements that cannot be identified by routine banding techniques. This approach is very important to complement the cytogenetic analysis of malignant disorders and to evaluate the role of chromosome change in the development, progression, and prognosis of tumors. Am. J. Hematol. 55:69-76, 1997. © 1997 Wiley-Liss, Inc.     

Paper chromatography hybridization: a rapid method for detection of Onchocerca volvulus DNA amplified by PCR

Prior studies have shown that Onchocerca volvulus DNA can be detected in skin snips and in black flies after polymerase chain reaction (PCR) with primers specific for repeated "O-150" DNA sequences. We have adapted a paper chromatography hybridization assay (PCHA) to detect amplified O-150 DNA and compared this method to two established methods, namely agarose gel electrophoresis (AGE) and hybridization enzyme-linked immunosorbent assay (ELISA). The minimum amounts of purified O-150 DNA detected by PCHA, AGE, and ELISA were 5, 10, and 2 ng, respectively. The three methods had similar estimated sensitivities for detecting O. volvulus DNA amplified from skin snips from African subjects with onchocerciasis (88%, 84%, and 91%, respectively). No false positive results were observed with skin snips from uninfected control subjects. The paper chromatography hybridization assay detects PCR products in 30 minutes without electricity or special equipment. This technology brings DNA detection a step closer to widespread use in field settings.     

Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization.

Comparative genomic hybridization was applied to 5 breast cancer cell lines and 33 primary tumors to discover and map regions of the genome with increased DNA-sequence copy-number. Two-thirds of primary tumors and almost all cell lines showed increased DNA-sequence copy-number affecting a total of 26 chromosomal subregions. Most of these loci were distinct from those of currently known amplified genes in breast cancer, with sequences originating from 17q22-q24 and 20q13 showing the highest frequency of amplification. The results indicate that these chromosomal regions may contain previously unknown genes whose increased expression contributes to breast cancer progression. Chromosomal regions with increased copy-number often spanned tens of Mb, suggesting involvement of more than one gene in each region.     

Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes.

We describe a technique for transferring electrophoretically separated bands of RNA from an agarose gel to paper strips. The RNA is coupled covalently to diazobenzyloxymethyl groups on the paper. After transfer and appropriate treatment of the paper to destroy remaining diazo groups, specific RNA bands can be detected by hybridization with 32P-labeled DNA probes followed by autoradiography. This procedure allows detection of specific RNA bands with high sensitivity and low background.     

High-resolution analysis of the human HLA-DR polymorphism by hybridization with sequence-specific oligonucleotide probes

The human major histocompatibility complex class II antigens of the HLA-D are highly polymorphic, surface proteins essential in the cellular interactions necessary for an immune response. The analysis of this polymorphism is crucial for (i) histocompatibility matching for transplantation and (ii) understanding the association between HLA-D and certain important diseases. The polymorphism of certain HLA-D haplotypes may escape detection by current methodologies. Analysis at the genomic level of the polymorphism of one of the HLA-D subregions HLA-DR, using oligonucleotide probes specific for the polymorphic regions, is capable of distinguishing single nucleotide differences. The DRw6 haplotype was analyzed in view of the lack of DRw6 specific sera. On the basis of nucleotide sequence analysis, the DRw6 haplotype consists of at least two subtypes. When analyzed with oligonucleotide probes, this split identifies new polymorphic groups that differ from the DRw6 serological subgroups.     

Mapping of single-copy DNA sequences on human chromosomes by in situ hybridization with biotinylated probes: enhancement of detection sensitivity by intensified-fluorescence digital-imaging microscopy

Two single-copy DNA segments of 6 kilobases (kb) and 2.3 kb were labeled with biotin-labeled dUTP (Bio11-dUTP) and hybridized to human chromosomes. These probes were detected by immunofluorescence and directly mapped on chromosomes by using classical fluorescence microscopy and a microchannel-plate-intensified video camera. By a subsequent R-banding, the 6-kb and 2.3-kb fragments were precisely localized to the 18p11.3 band and to the 22q11.2 band, respectively, in agreement with previous results obtained with radioactive probes. The adaptation of fluorescence intensification and digital image processing (frame integration to enhance signal-to-noise ratio and linear contrast stretching) to microscopy makes it possible to detect very weak fluorescent spots on chromosomes. This system allows a high spatial resolution (less than 0.6 micron), even at very low fluorescence levels. The efficiency and the specificity of the hybridization and detection methodology give a direct and precise localization of the short single-copy sequences on human chromosomes.     

Human papillomavirus detection in cervical dysplasias or neoplasias and in condylomata acuminata by in situ hybridization with biotinylated DNA probes.

Specimens from cervical dysplasias or carcinomas and genital condylomata acuminata were retrospectively analysed by in situ hybridization (ISH) with biotinylated DNA probes for human papillomavirus (HPV) types 6, 11, 16 and 18. In the control group no case was positive for HPV DNA. In mild/moderate dysplasias, 4 cases (14%) were positive for HPV 6 or 11 and 2 cases (7%), for HPV 16. In the severe dysplasia/in situ carcinoma group, 9 cases (31%) showed presence of DNA of HPV types 16 or 18. Six invasive carcinomas (20%) were positive for HPV type 16 or 18. Among condylomata acuminata, 22 cases (73%) were positive for HPV types 6 or 11. In all ISH-positive cases only one viral type was detected. No correlation between HPV DNA positivity and histological findings of HPV infection was observed. Although less sensitive than some other molecular biology techniques, in situ hybridization with biotinylated DNA probes proved to be simple and useful for detecting and typing HPV in samples routinely received for histopathological analysis.     

Targeted correction of the point mutations of beta-thalassemia and targeted mutagenesis of the nucleotide associated with HPFH by RNA/DNA oligonucleotides: potential for beta-thalassemia gene therapy

An RNA/DNA chimeric oligonucleotide was found to be effective in the targeted correction of point mutations in Escherichia coli, plant, and mammalian genomes. This strategy, named chimeraplasty, has the potential for gene therapy of many genetic diseases caused by point mutations. beta-Thalassemia is a very common human genetic disease and in most cases it is caused by point mutations. To test whether the chimeraplasty can be used to correct the point mutations responsible for beta-thalassemia, we introduced one mutated beta-globin gene, betaE, into MEL cells and successfully corrected the point mutation of the betaE gene with the highest correction efficiency of 1.9%. Furthermore, a targeted -202 C-->G mutation of the Ggamma-globin gene, which is associated with the elevated Ggamma-globin gene expression in the adult stage, was introduced into HeLa and CMK cells by an RNA/DNA oligonucleotide. These results indicated that the chimeraplasty has potential for human beta-thalassemia gene therapy.     

Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy.

Combinatorial labeling of probes (i.e., with two or more different reporters) increases the number of target sequences that can be detected simultaneously by fluorescence in situ hybridization. We have used an epifluorescence microscope equipped with a digital imaging camera and computer software for pseudocoloring and merging images to distinguish up to seven different probes using only three fluorochromes. Chromosome-specific centromere repeat clones and chromosome-specific "composite" probe sets were generated by PCR in which different mixtures of modified nucleotides, including fluorescein-conjugated dUTP, were incorporated. Cosmid clones were labeled similarly by nick-translation. The technique has been used to delineate the centromeres of seven different human chromosomes, on both 4',6-diamidino-2-phenylindole-stained metaphase spreads and interphase nuclei, to map six cosmid clones in a single hybridization experiment and to detect chromosome translocations by chromosome painting. Multiparameter hybridization analysis should facilitate molecular cytogenetics, probe-based pathogen diagnosis, and gene mapping studies.     

A review of cis-trans interplay between DNA sequences 5' to the (G)gamma- and beta-globin genes among Hb F-Malta-I heterozygotes/homozygotes and beta-thalassemia homozygotes/compound heterozygotes, and the effects of hydroxyurea on the Hb F/F-erythrocyte; the need for large multicenter trials

The biosynthesis of Hb F in place of the deficient Hb A could be a suitable treatment for beta hemoglobinopathies. Among newborn Hb F-Malta-I heterozygotes, it could be shown that the XmnI sequence alone had little, if any effect on gamma-globin gene expression, but interplay with the (AT)(X)T(Y) sites in cis and in trans may occur. In contrast, while the XmnI sequence is clearly correlated with gamma-globin levels in anemic adult beta-thalassemia (thal) homozygotes, the effect on F-erythrocyte numbers and Hb F/F-erythrocyte appears independent of the (AT)(X)T(Y) sites. Even at levels of hydroxyurea (HU) as low as 1.65 mg/kg/day (vs. 10 mg/kg/day on the high dose regime) it can be shown that although even a small increase of Hb F could be obtained, the effect was rarely translated into an increase in circulating hemoglobin (Hb). In most cases, the elevated Hb F level was dependent on the XmnI sequence and was due to increased numbers of F-erythrocytes or Hb F/F-erythrocyte or both. It seems that the bone marrow of thalassemia homozygotes may be more sensitive to myelosuppression by HU possibly due to medullary inflammation. While the data are consistent with loop models of globin switching mechanisms, there is urgent need for large, hypothesis driven, multicenter trials of molecules that could maintain or re-induce high Hb F levels in beta-thal and subject to genetic and epigenetic constraints including inflammation.     

Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes

Background

Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damnage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid.

Methods

Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity.

Results

Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii.

Conclusion

We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis.

     

Localization by situ hybridization of amplified ribosomal DNA during Xenopus laevis oocyte maturation (a light and electron microscopy study).

The presence of ribosomal DNA has been demonstrated, by light and electron microscopy study of in situ hybridization with 125I-labeled ribosomal RNA, in the Feulgen-positive bodies which appear during maturation in the cytoplasm of Xenopus laevis oocytes. The ultrastructure of these bodies is described.