Biomedical subjects

Ju Yan

Publications and source records attributed to Ju Yan.

7 recordsLinked to original sources

PubMed2006

Dual-color PRINS for in situ detection of fetal cells in maternal blood.

Fetal nucleated cells circulating in the peripheral blood during pregnancy are potential targets for noninvasive genetic testing. Fluorescence in situ hybridization (FISH) frequently is used to quantify the total number of fetal cells in peripheral blood of pregnant women. We describe an alternative molecular cytogenetic procedure that is the primed in situ labeling (PRINS). This technique consists of annealing oligonucleotides specific to individual chromosome targets and in situ elongation using Taq DNA polymerase to incorporate labeled dUTPs. The sites of the newly synthesized DNA sequences were revealed as fluorescent signals using an immunochemical reaction. The dual-color PRINS was specifically performed for simultaneous detection of two chromosome targets, X and Y. The fluorescent signals corresponding to chromosomes X and Y were displayed as red and green color spots, respectively. The sensitivity and specificity of PRINS are similar to FISH and allow us to efficiently and reliably detect fetal cells in maternal blood. Moreover, dual-color PRINS is faster and more cost-effective than FISH.

Chromosomes, Human, X↗

PubMed2006

New rapid multicolor PRINS protocol.

In the multiple-color primed in situ labeling (multi-PRINS) technique, using ddNTPs between two PRINS reactions can block the free 3'-end generated in the previous PRINS reaction, thus avoiding the next PRINS reaction, using it as a primer to perform spurious elongation at nondesired sites. However, by omitting the blocking step and taking advantage of the color mixing, we developed a simple and rapid multi-PRINS technique to simultaneously detect three chromosomes in the same cell. With this protocol, one can create a third color using the two most common forms of labeled dUTP (biotin- and digoxigenin-labeled dUTP) and two fluorochromes (fluorescein and rhodamine). The signals at the centromeres of three different chromosomes displayed perfect yellow, red, and green colors, respectively. The entire procedure could be completed in less than 90 min because the blocking step was omitted. This protocol is practical and efficient for multi-PRINS so that even more than three chromosome targets could be detected in the same cell.

Chromosomes, Human↗

PubMed2004

The labeling efficiency of human telomeres is increased by double-strand PRINS.

Telomeres are composed of tandem repeated sequences, TTAGGG, that can be detected either by fluorescence in situ hybridization (FISH), more efficiently by using a peptide nucleic acid (PNA) probe, or by the primed in situ (PRINS) technique. However, the efficiency of human telomere labeling using PRINS is somewhat lower than the efficiency using PNA-FISH. To solve this problem, we developed a double-strand PRINS technique, which uses two primers, (TTAGGG)(7) and (CCCTAA)(7), to label both forward and reverse telomeric DNA strands. A total of 120 lymphocyte metaphases obtained from three normal adults were scored to evaluate the labeling efficiency based upon the telomere signal frequency present in chromatid ends and chromosome arms. As a comparison, 30 metaphases from the same three individuals were evaluated using PNA-FISH. The average labeling efficiency of PRINS was increased to a level very close to that obtained with PNA-FISH. Therefore, we demonstrated that the low labeling efficiency of human telomeres with regular PRINS was likely caused by uneven annealing of primers at the relatively short human telomere sequences, resulting in some telomere sites with very weak or absent labeling. We suggest that the present double-strand labeling protocol is critical to maximize the labeling efficiency of the human telomere sequence when using the PRINS technique.

Adult↗

PubMed2004

[From the conception of the PRINS to its coronation].

As a non-isotopic molecular cytogenetic technique, the primed in situ (PRINS) labelling reaction represents a major technological progress achieved in the past decade. It has become a routine technique for the microscopic visualization of specific DNA sequences in cells and nuclei and constitutes a good alternative to the fluorescence in situ hybridization (FISH) procedure. Among the multiple advantages that characterize the PRINS technique, specificity, rapidity, reliability, reproducibility, and cost-effectiveness can be mentioned. PRINS can be in addition associated with other techniques like FISH, indirect immunofluorescence, and nick translation. The most recent developments show the great potential of this technique. Now PRINS can be used to study single-copy genes and, consequently, can be routinely used to investigate deletions associated with microdeletion syndromes. Therefore, the PRINS technique has the potential to become a widely used molecular cytogenetic tool in clinics and research. This short review presents how the PRINS technique contributed to further the understanding of biological phenomena and describes the different possibilities and applications of the PRINS method in several biological and clinical fields (pre-implantation testing, prenatal, constitutional and oncologic genetic diagnosis).

Base Sequence↗

PubMed2003

A unique clone involving multiple structural chromosome rearrangements in a myelodysplastic syndrome case.

In a young female patient presenting with a myelodysplastic syndrome (MDS), a unique clone involving six structural chromosome rearrangements was identified using G-banding and molecular cytogenetic techniques. Fifty GTG-banded metaphases from bone marrow were initially analyzed and all metaphases contained all of the six structural chromosome rearrangements. To further define the GTG-banded karyotype, a series of fluorescence in situ hybridization and primed in situ labeling experiments were performed and the karyotype was then characterized as: 46,XX,r(5)(p13q13),der(20)t(5;20),dup(11)(p11.2p15), r(11)(p15q25),del(13)(q14),idic(22)(p11). The patient quickly progressed to acute nonlymphocytic leukemia three months after the diagnosis and died of a hemorrhage in the brain parenchyma two months later. In this case, the multiple structural chromosome rearrangements conferred an obvious cellular proliferative advantage and indicated a very poor prognosis. Considering that multiple chromosome abnormalities associated with MDS transformation are often polyclonal, this unique clone involving six structural chromosome rearrangements make our case highly unusual.

Adult↗

PubMed2002

Evidence that extrachromosomal double-strand break repair can be coupled to the repair of chromosomal double-strand breaks in mammalian cells.

Transfected linear DNA molecules are substrates for double-strand break (DSB) repair in mammalian cells. The DSB repair process can involve recombination between the transfected DNA molecules, between the transfected molecules and chromosomal DNA, or both. In order to determine whether these different types of repair events are linked, we devised assays enabling us to follow the fate of linear extrachromosomal DNA molecules involved in both interplasmid and chromosome-plasmid recombination, in the presence or absence of a pre-defined chromosomal DSB. Plasmid-based vectors were designed that could either recombine via interplasmid recombination or chromosome-plasmid recombination to produce a functional beta-galactosidase (betagal) fusion gene. By measuring the frequency of betagal+ cells at 36 h post-transfection versus the frequency of betagal+ clones after 14 days, we found that the number of cells containing extrachromosomal recombinant DNA molecules at 36 h (i.e., betagal+), either through interplasmid or chromosome-plasmid recombination, was nearly the same as the number of cells integrating these recombinant molecules. Furthermore, when a predefined DSB was created at a chromosomal site, the extrachromosomal recombinant DNA molecules were shown to integrate preferentially at that site by Southern and fiber-FISH (fluorescence in situ hybridization) analysis. Together these data indicate that the initial recombination event can potentiate or commit extrachromosomal DNA to integration in the genome at the site of a chromosomal DSB. The efficiency at which extrachromosomal recombinant molecules are used as substrates in chromosomal DSB repair suggests extrachromosomal DSB repair can be coupled to the repair of chromosomal DSBs in mammalian cells.

Animals↗

PubMedDate not supplied

The telomere length dynamic and methods of its assessment.

Human telomeres are composed of long repeating sequences of TTAGGG, associated with a variety of telomere-binding proteins. Its function as an end-protector of chromosomes prevents the chromosome from end-to-end fusion, recombination and degradation. Telomerase acts as reverse transcriptase in the elongation of telomeres, which prevent the loss of telomeres due to the end replication problems. However, telomerase activity is detected at low level in somatic cells and high level in embryonic stem cells and tumor cells. It confers immortality to embryonic stem cells and tumor cells. In most tumor cells, telomeres are extremely short and stable. Telomere length is an important indicator of the telomerase activity in tumor cells and it may be used in the prognosis of malignancy. Thus, the assessment of telomeres length is of great experimental and clinical significance. This review describes the role of telomere and telomerase in cancer pathogenesis and the dynamics of the telomeres length in different cell types. The various methods of measurement of telomeres length, i.e. southern blot, hybridization protection assay, fluorescence in situ hybridization, primed in situ, quantitative PCR and single telomere length analysis are discussed. The principle and comparative evaluation of these methods are reviewed. The detection of G-strand overhang by telomeric-oligonucleotide ligation assay, primer extension/nick translation assay and electron microscopy are briefly discussed.

Aging↗