Antti Iitiä

Determination of a common genetic variant of luteinizing hormone using DNA hybridization and immunoassays.

An immunologically anomalous form of LH, due to two point mutations in codons 8 and 15 of the LHβ gene, has previously been described. LH status, i.e. the discrimination between wild‐type (WT) and variant (V) LH, is usually determined by immunoassays, which can be unreliable at low serum concentrations of LH. A DNA hybridization assay was therefore developed to score the LH genotype in all subjects, independent of their serum LH concentrations. To evaluate the performance of the hybridization method, and to expand our observations of the worldwide occurrence of the V‐LH, we determined its frequency in additional populations. To confirm the connection between the anomalous immunoreactivity and the V‐LHβ gene, we also sequenced the LHβ subunit gene of a homozygous person.

The use of europium (Eu3+) labelled primers in PCR amplification of specific target DNA

The polymerase chain reaction (PCR) has many potential applications in the field of DNA probe diagnostics. Here we describe a method that utilizes PCR and time-resolved fluorometry (TRF) for the detection of specific target DNA. First the DNA segment to be detected is amplified according to standard procedures. Then a pair of europium (Eu3+) and biotin-labelled primers nested within the amplified fragment is incorporated in a few additional PCR cycles. Thus amplified DNA fragments are generated that contain an affinity label (biotin) and a detectable label (europium). The doubly-labelled amplified DNA fragments are collected onto streptavidin coated microtitration strips and the bound Eu3+ is measured in a time-resolved fluorometer. We show here the application of this method to the detection of HIV-1 DNA. As few as five copies of HIV-1 DNA could readily be detected using this assay. The method described here is sensitive, rapid and easy to employ. In addition it lends itself to automation.

Detection of amplified HTLV-I/II viral sequences using time-resolved fluorometry

Since its discovery, the polymerase chain reaction (PCR) has been used for different purposes in the field of DNA research. We tested the PCR for the diagnosis of HTLV-I/-II infections. PCR was used to amplify 141- and 149-base pair regions from the HTLV-I and HTLV-II virus genomes, respectively. The annealing temperature in the PCR amplification was optimized using 20% polyacrylamide gels and silver staining. Even a slight change (3 degrees C) in the annealing temperature had an effect on the specificity of the reaction. The PCR products were detected with biotin and Eu-labeled oligonucleotide probes in a solution hybridization format. The linearity of the assay was tested with serial dilutions of purified chromosomal DNA containing integrated HTLV-II sequences. The linearity was found to be dependent on the number of cycles used in the PCR amplification. The best linearity, at a target level of a few copies, was achieved using a low number of cycles. The specificity of the assay was tested using HTLV-I and HTLV-II-infected lymphocytes from the cell lines Hut102 and MO480, respectively. No cross reactivity between these analytes was observed.

Detection of Philadelphia chromosome using PCR and europium-labeled DNA probes.

More than 95% of the patients with chronic myelogenous leukemia (CML) carry translocations between protooncogene abl of chromosome 9 and bcr gene of chromosome 22, resulting in the Philadelphia chromosome (Ph1). After allogeneic bone marrow transplantation (BMT) it is important to detect possible residual malignant cells in CML patients. A new sensitive hybridization method combined with polymerase chain reaction (PCR), based on the detection of the europium (Eu3+) label by time-resolved fluorescence, was applied for the detection of Ph1 chromosome. Total RNA from 10(6) peripheral blood leukocytes was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction. After cDNA synthesis by reverse transcriptase, the PCR amplification (30 cycles) was carried out. In the detection phase two oligonucleotide probes were used in the hybridization reaction, one biotinylated (bcr gene, exon 2) and one (abl gene) labeled with Eu3+. The hybrids were collected in a streptavidin-coated microtitration well and the bound Eu3+ was measured in a time-resolved fluorometer. To assess the sensitivity of the method, different numbers of CML cell line K562 cells were mixed with 10(5) apparently normal human leukocytes. Five K562 cells/10(5) leukocytes could be detected. Six patients with CML confirmed by clinical and cytogenetic criteria were studied. Three of the patients underwent an allogeneic BTM 6-18 months before the investigation and all of them were Ph1-negative. The other three patients who were nontransplanted were positive as expected.