Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques

ABSTRACT Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.

Rapid detection of chromosome X, Y, 13, 18, and 21 aneuploidies by primed in situ labeling/synthesis technique.

AIMS AND OBJECTIVE: Primed in situ labeling/synthesis (PRINS) technique is an alternative to fluorescent in situ hybridization for chromosome analysis. This study was designed to evaluate the application of PRINS for rapid diagnosis of common chromosomal aneuploidy. MATERIALS AND METHODS: We have carried out PRINS using centromere specific oligonucleotide primers for chromosome X, Y, 13, 18 and 21 on lymphocyte metaphase and interphase cells spread. Specific primer was annealed in situ, followed by elongation of primer by Taq DNA polymerase in presence of labeled nucleotides. Finally, reaction was stopped and visualized directly under fluorescent microscope. RESULTS: Discrete centromere specific signals were observed with each primer. CONCLUSION: PRINS seems to be a rapid and reliable method to detect common chromosome aneuploidy in peripheral blood lymphocyte metaphase and interphase cells.

Exequibility of differential gene expression analysis by DDRT-PCR in murine bone marrow cells / Exequibilidade do DDRT-PCR para análise da expressão gênica diferencial em células de medula óssea murina

Model of study: Experimental study. Introduction: Recently, stem cell research has generated great interest due to its applicability in regenerative medicine. Bone marrow is considered the most important source of adult stem cells and the establishment of new methods towards gene expression analysis regarding stem cells has become necessary. Thus Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR) may be an accessible tool to investigate small differences in the gene expression of different stem cells in distinct situations. Aim: In the present study, we investigated the exequibility of DDRT-PCR to identify differences in global gene expression of mice bone marrow cells under two conditions. Methods: First, bone marrow cells were isolated fresh and a part was cultivated during one week without medium replacement. Afterwards, both bone marrow cells (fresh and cultivated) were submitted to gene expression analyses by DDRT-PCR...

Modelo do estudo: Estudo Experimental. Introdução: Atualmente a pesquisa com células-tronco tem gerado grande interesse devido a sua aplicabilidade no campo na medicina regenerativa. A medula óssea é considerada a maior fonte de células-tronco adultas e o estabelecimento de novos métodos para a análise da expressão gênica torna-se estritamente necessário. Desse modo, o "Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR)", pode ser uma ferramenta acessível para investigação de pequenas diferenças no nível de expressão gênica em diferentes tipos celulares, sob distintas condições. Objetivo: Neste presente trabalho nós investigamos a exequibilidade do DDRT-PCR na identificação de diferenças no nível de expressão gênica global em células da medula óssea de camundongos sob duas condições. Métodos: Primeiramente, a medula óssea foi isolada frescamente e uma secunda parte foi cultivada por uma semana sem troca de meio. Posteriormente, as células da medula (fresca e cultivada) foram submetidas a análise da expressão gênica, seguindo a metodologia de DDRT-PCR...

Rapid detection of SOX2 gene by primed in situ labeling / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To rapidly detect SOX2 gene using primed in situ labeling (PRINS).</p><p><b>METHODS</b>Human peripheral blood samples were cultured using an optimized method. Sequence of the SOX2 gene was amplified in situ with biotin-labeled specific primers and processed with a tyramide signal amplification (TSA) biotin system. Subsequently, fluorescence-stained signal was detected by streptavidin-Texas red. For the control group, MCF-10F cells were transfected with Lentivirus hSox2.</p><p><b>RESULTS</b>By VideoTesT-FISH software analysis, the long arm of chromosome 3 in the experimental group showed a specific red fluorescence signal, whilst the control samples showed no specific signals for SOX2. Transfected MCF-10F cells showed various efficiency of SOX2 gene integration.</p><p><b>CONCLUSION</b>PRINS utilizes a highly sensitive in situ PCR technique combined with fluorescence labeled oligodeoxynucleotides can synthesize probes in situ, thus greatly reducing the cost of probe and time for detection. It can facilitate identification and classification of induced pluripotent stem cells, and has many potential applications in this prospect.</p>

Application of multicolor primed in situ labeling in simultaneous identification of chromosomes 18, X and Y in uncultured amniocytes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a multicolor primed in situ labeling (PRINS) protocol for chromosome detection in uncultured amniocytes.</p><p><b>METHODS</b>Chromosomes 18, X and Y in uncultured amniocytes were simultaneously detected by using the non-ddNTP-blocking multicolor PRINS procedure.</p><p><b>RESULTS</b>Within 7 h, the 3 chromosomes were simultaneously marked in the same uncultured amniocyte. The chromosome signals were successfully detected in 69 uncultured samples of amniotic fluid. The results were consistent with that obtained by chromosomes in cultured amniocytes.</p><p><b>CONCLUSION</b>This multicolor protocol was high throughput, fast, simple, sensitive and reliable in diagnosing chromosome abnormalities in uncultured amniocytes.</p>

Detection of the SRY gene by primed in situ labeling / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a primed in situ labeling (PRINS) technique which can be more effective in detection of single copy gene.</p><p><b>METHODS</b>On the basis of traditional PRINS, new reagents and procedures, such as TaqStart antibody, four primers of the sex determining region Y (SRY) gene and TSA(TM) Biotin System were included in detection of the SRY gene. Meanwhile, fluorescence in situ hybridization(FISH) to detect the SRY gene was used as control.</p><p><b>RESULTS</b>Fifty metaphases were scored. PRINS labeling showed signals for the SRY on the Y chromosome at band Yp11.3 in all metaphases. These signals were as distinct as that from results of FISH.</p><p><b>CONCLUSION</b>This improved method is ideal for rapidly localizing single copy genes and small DNA segments. And PRINS is a cost- and time-effective alternative to FISH.</p>

Rapid detection of chromosomes X and Y using dual-color primed in situ labeling technique / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a rapid method for detection of chromosomes, and to present a feasible method for rapid detection of the interphase nuclei of uncultured amniocytes using modified dual-color primed in situ labeling (PRINS) technique.</p><p><b>METHODS</b>Chromosomes X and Y were detected and analyzed by the modified dual-color PRINS in 205 amniotic fluid samples.</p><p><b>RESULTS</b>The specific chromosomes were obtained on both metaphase and interphase nuclei. In more than 98% of the samples PRINS reactions with primers X and Y were successfully induced. One sample was properly identified and correctly scored as 47, XXY.</p><p><b>CONCLUSION</b>The results suggest that PRINS technique is rapid, simple and cost-effective. It is also sensitive and specific, and thus useful for rapid detection of chromosome abnormalities.</p>

Triple-color primed in situ labeling protocol of human metaphase chromosomes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the feasibility of simultaneous detection for several chromosomes with optimized triple-color primed in situ labelling (PRINS) protocol in cultured peripheral blood lymphocytes.</p><p><b>METHODS</b>Pre-test of gonosome detection with dual-color PRINS protocol was performed to explore and optimize the order and condition of PRINS primers. A peripheral blood sample from a Klinefelter's syndrome patient (47, XXY) had also been studied with optimized triple-color PRINS to prove the correspondence between the number of signals and chromosomes.</p><p><b>RESULTS</b>Chromosome 18, X and Y had been simultaneously and specifically marked within 3 hours. The frequency of successful labeling reached 90% both in dual-color and triple-color test. Two chromosome X had been correctly showed in lymphocyte sample of Klinerfelter's syndrome.</p><p><b>CONCLUSION</b>Numerical chromosome anomalies could be rapidly and exactly detected with this non-ddNTP-blocking multicolor PRINS protocol in peripheral blood lymphocytes. The results of in situ labeling are much clearer with inner control.</p>

In situ aneuploidy assessment in human sperm: the use of primed in situ and peptide nucleic acid-fluorescence in situ hybridization techniques / 亚洲男科学杂志(英文版)

Both the primed in situ (PRINS) and the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) techniques constitute alternatives to the conventional (fluorescence in situ hybridization, FISH) procedure for chromosomal investigations. The PRINS reaction is based on the use of a DNA polymerase and labeled nucleotide in an in situ primer extension reaction. Peptide nucleic acid probes are synthetic DNA analogs with uncharged polyamide backbones. The two procedures present several advantages (specificity, rapidity and discriminating ability) that make them very attractive for cytogenetic purposes. Their adaptation to human spermatozoa has allowed the development of new and fast procedures for the chromosomal screening of male gametes and has provided efficient complements to FISH for in situ assessment of aneuploidy in male gametes.

Rapid detection of 18-trisomy syndrome using primed in situ labeling technique / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a rapid method for the detection of chromosomes and try to verify the feasibility of using modified primed in situ labeling (PRINS) technique for rapid detection of the interphase nuclei of uncultured amniocytes.</p><p><b>METHODS</b>Chromosome 18 was detected and analyzed by the modified PRINS in 262 amniotic fluid samples.</p><p><b>RESULTS</b>The specific chromosomes were obtained on both metaphase and interphase nuclei. In more than 95% of the samples, PRINS reactions with primer 18cen were successfully induced. Two samples were properly identified and correctly scored as trisomic 18.</p><p><b>CONCLUSION</b>The results suggest that PRINS technique is simple, rapid and cost-effective. It is sensitive, specific, and thus can enhance the accuracy of standard cytogenetic analysis.</p>

Preimplantation genetic diagnosis / 中华医学遗传学杂志

Preimplantation genetic diagnosis is a very early form of prenatal diagnosis aimed at eliminating embryos carrying serious genetic diseases before implantation. The basic techniques currently used involve embryo biopsy, the polymerase chain reaction and fluorescence in situ hybridization. In the current review, a number of problems arising from the use of these technologies as well as the possible solutions and new developments are discussed.

Diagnostico genetico pre-implantacional / Polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and primed in situ (PRINS)

O diagnostico genetico pre-implantacional (PGD) e um novo procedimento que pode ser utilizado como diagnostico precoce pre-natal em casais com risco de transmissao de doencas geneticas. Utilizando "polymerase chain reaction" (PCR), "fluorescence in-situ hybridization" (FISH) e "primed in-situ" (PRINS) pode-se determinar o genotipo ou sexo genetico do embriao biopsiado obtido por...

Clinical use cytogenetic karyotyping , fluorescence in situ hybridization , and primed in situ labelling in prenatal diagnosis / 대한산부인과학회잡지

OBJECTIVE: Increasingly it is being recognized that genetic factors play a significant role in causing malformation. There are many available prenatal diagnostic methods including cytogenetic karyotyping using amniocentesis and cordocentesis, fluorescence in situ hybridization(FISH), and primed in situ labelling(PRINS). Our purpose was to attempt to discuss the clinical use of cytogenetic karyotyping, FISH, and PRINS. METHODS: We conducted 222 cases of cytogenetic karyotyping using amniocentesis and cordocentesis, l0 cases of FISH, and 10 cases of PRINS from January 1996 to July 1998 at Ewha Womans University Mokdong Hospital. Age distribution, chromosomal abnormalities by age group, indication, karyotype, and baby outcomes were performed. RESULTS: Overall incidence of chromosomal abnormalities was 7.7%(17cases) and chromosomal abnormalities were most frequently noted in 30-34 year old women and 35-39 year old women(2.3%, respectively). Among 222 cases, 25-29 year old women were highest(30.2%). Chromosomal abnormalities among cytogenetic karyotyping cases were Down syndrome, Edward syndrome, Patau syndrome, Deletion(8), Inversion(9), etc. The 5 cases of healthy baby among chromosomal abnormalities were delevered. Among 213 cases of karyotyping using amniocentesis, abnormal karyotyping cases were 15 cases. Among 15 cases, 8 cases were terminated and 5 cases of healthy baby were delivered. Among 9 cases of karyotyping using cordocentesis, 2 cases of chromosomal abnormalities(Edward, Down syndrome) were found and 3 cases healthy baby were delivered. Among 10 cases of FISH results, 6 case of FISH results were the same with G-banding and were different from G-banding. Among 10 cases of PRINS results, we got the PRINS results from 7 cases. CONCLUSION: It is concluded that cytogenetic karyotyping, FISH, and PRINS are very useful to detect chromosomal abnormalities.

Study on the Detection of Chromosome X, Y Using in situ PCR / 대한산부인과학회잡지

Untill now, classical karyotyping remains the most reliable and conclusive method, but sometimes very rapid and sensitive method for the detection of chromosomal abnormality is needed. The primed in situ labelling(PRINS) is very rapid and sensitive method for the detection of chromosomal aneuploidy and X-linked disorder instead of fluorescen in situ hybridization. The purpose of this study was to prepent the application of PRINS protocol to the rapid detection of chromosomes X and Y in both metaphases and interphase nuclei. The PRINS reaction was done in 10 amniocytes. We compared the PRINS result with conventional cytogenetic karyotyping. The result was the same with cytogenetic karyotyping. In the future, PRINS will become very useful and sensitive method for the chromosomal aneuploidy detection and X-linked disorder diagnosis.