Preparation of Samples

Drop the cell suspension onto precleaned slides. Thermotron conditions should be 25 C and 50 humidity (see Notes 4 and 5). 2. Incubate the slides in 2X SSC for 15 to 30 min at 37 C and then dehydrate the slides through a series of ethanol (70 , 80 , and 100 ) at room temperature. For amniotic cells, a specific pretreatment is applied (see Note 6). 3. Prepare 50 mL of denaturation solution (70 formamide 2X SSC) by mixing thoroughly 35 mL of formamide (ultrapure grade, Fluka), 5 mL of 20X SSC,...

PRINS Reaction

Glass slides (25 x 75 x 1 mm VWR Scientific, Inc.) and selected micro cover glasses, cover slips (24 x 30 mm) (VWR Scientific, Inc.). The slides must be cleaned by soaking in series of ethanol solutions followed by polishing with a clean piece of muslin and wiped dry using lint-free paper before dropping the cells on the slides. 2. Variable volume micropipets ranging from 0 pL to 1000 pL. 3. Microcentrifuge tubes (1.5 mL). 4. Plastic and glass Coplin jars, or other suitable containers for...

Application of Molecular Cytogenetics to the Micronucleus Assay

Because micronuclei originating from the two pathways depicted in Fig. 1 are morphologically identical, the preferential mechanism of action (i.e., clastogenic vs aneuploidogenic activity) of many agents of environmental importance cannot be directly defined. With the advent of molecular cytogenetics, different strategies were used to distinguish between micronuclei originating from chromosome breaks or chromosome loss the key DNA sequences are the centromeric and telomeric sequences because...

The Micronucleus Assay

In mammalian cells, small bodies of extra-chromatin denoted micronuclei can be observed in the cytoplasm at interphase and represent a rare abnormality consequent to genetic damage. Micronuclei are formed at the mitotic division by two different pathways (Fig. 1) (1) a pre-existing chromosome fragment lacking the centromere and, therefore, unable to move toward the spindle poles, can decondense into a micronucleus if excluded from the daughter nuclei (Fig. 1A) or (2) micronuclei can be derived...

Hybridization

Prepare a solution containing 20-50 pg pL of the appropriate probe, 50 deionized formamide, 2X SSC buffer, 10X Denhardt's solution, 0.1 sonicated ssDNA, and 0.1 SDS. The following is a convenient recipe for a total volume of 100 pL (see Note 20) 1. Add 10 pL of hybridization mixture to each well and add cover slips. 2. Heat slides on a block at 95 C for 5 min. 3. Incubate slides at 48 C for 2 to 4 h in a humidified atmosphere. 3.7. Posthybridization for Peroxidase-Based Color Development 1....

Primed In Situ Labeling Standard Protocol

Immerse slides in 0.02 NHCl for 20 min (see Note 6). 2. Immerse slides in 70 formamide-SSC, pH 7.0 for 2 min at 72 C, to denature chromosomal DNA. 3. Dehydrate slides by passage through a cold ethanol series, 70 , 85 , and 100 EtOH, 5 min each. Air-dry. 4. Prepare reaction mixture in a final volume of 40 pL containing 50 pmol of each oligonucleotide primer (see Note 7) 0.2 mMeach dATP, dCTP, dGTP 0.02 mM dTTP 0.02 mM biotin-16-dUTP 50 mM KCl 10 mM Tris-HCl, pH 9.0 2 mM MgCl2 0.01 BSA and 1 unit...

PRINS Reactions

Mix 5 L of primer stock solution with 95 L of TE buffer or water for a 5 M primer working solution. 2. For each slide, mix in a 1.5-mL microtube b. 4 L of each dNTP working solution, a total of 16 L. c. 2 L of primer working solution. d. 1 L of 1 mMbiotin-dUTP or 1 L of 1 mM dig-dUTP. e. 2.5 L of glycerol (see Note 7). g. 0.5 L of Taq DNA polymerase (add immediately before starting the PRINS reaction). 1. Initiate the PRINS reaction by adding the first reaction solution (50 L) containing a...

Introduction

Microdissection is an extremely useful method of precisely isolating chromosomes, chromosome fragments, and bacteria (1-3). With few exceptions, microdissection has required that multiple copies of a desired template be isolated, to obtain sufficient material for subsequent amplification. This is frequently possible, as in the case of G-banded human chromosomes. However, for many case, such as isolated fragments, specific bands, and derivative chromosomes, it is advantageous to be able to...

Avirachan T Tharapel and Stephen S Wachtel

The chromosomal regions 13q14 and 17p13 often are found rearranged in hematopoietic tumors in humans, but the rearrangements can be subtle and can escape detection on gross cytogenetic analysis. For example, submicroscopic perturbations of the RB1 and p53 tumor suppressor genes, located in 13q14 and 17p13, respectively, frequently occur in leukemias this has been confirmed by molecular methods such as fluorescence in situ hybridization (FISH). Our group modified the primed in situ labeling...

Basic Ingredients and Instrumentation

Chromosome spreads (preferably freshly made see Note 1). 3. Coplin jars or other suitable containers for washing the slides. 4. Standard nucleoside triphosphates, dideoxy nucleotides (Lithium salt, e.g., Roche). 5. Hapten- or fluorochrome-labeled nucleotides (digoxigenin-2'-deoxyuridine 5'-triphosphate dUTP , biotin-dUTP, fluorescein-dUTP, rhodamine-dUTP, etc. e.g., Roche). 7. Thermostable DNA polymerase that will accept labelled nucleotides (e.g., Tth or Taq DNA polymerase) and 10X polymerase...

Indirect Immunofluorescence

First antibody anti-P-tubulin antibody (monoclonal from mouse Sigma see Table 1). Antibodies and Fluorescent Reagents Used in the Study Antibodies and Fluorescent Reagents Used in the Study Observation Systems Used For Acquisition With the Confocal Microscope Observation Systems Used For Acquisition With the Confocal Microscope 2. Second antibody tetramethylrhodamine isothiocyanate (TRITC)-labeled anti-mouse F(ab')2 fragments (from donkey) (Jackson Immunoresearch, West Grove, PA see Table 1)....

Methods

In essence, the PRINS method consists of 4 steps (1) DNA denaturation, (2) annealing of primers, (3) chain extension in the presence of labeled nucleotides and Taq DNA polymerase, and (4) detection of newly-synthesized labeled DNA by use of antibodies complexed to fluorescent dyes. The oligo-nucleotide primers are 16 to 30 base pairs in length. A typical reaction mixture for PRINS has a volume of 40 L and contains 200 to 500 pmol of primer (see Note 4). The reaction mixture is placed on the...

Preparation of Interphase Nuclei Suspension From the Specimens of Fetal and Adult Brain Tissues

Taped homogenizer Teflon pestle and glass tube (Cole-Parmer International cat. nos. A-04368-32 for Teflon pestle and A-04368-33 for glass tube). 2. Earle's buffered saline solution (EBBS cat. no. 24010-035, Gibco Invitrogene, SARL Cergy Pontois Cedex, France). 3. Solution of phosphate-buffered saline (PBS) pH 7.3, containing 0.1 (w v) of Nonidet P-40. PBS preparation prepare 10X stock water solution with 1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, and 18 mM KH2PO4 (pH 7.4 is adjusted by 1 N HCl)....

Contributors

Yoshihiro Adachi Department of Pathology, Kochi Medical School, Kochi, Japan Tal Anahory Department of Reproductive Biology B, CHU Arnaud de Villeneuve, Montpellier, France Brigitte Andr o Institute of Human Genetics, CNRS UPR, Montpellier, France Omar Bagasra Department of Biology, Claflin University, Orangeburg, SC Allen T. Christian Biosciences Division, Lawrence Livermore National Laboratory, Livermore, CA Caterina Cinti Institute of Clinical Physiology, CNR, Siena Unit, Siena, Italy...

Signal Amplification Using the TSA Biotin System

For each test, dilute the stock solution of biotinyl tyramide 1 50 with 1X amplification diluent to prepare the working solution 100 to 300 pL of working solution are needed for each slide. 2. After hybridization, block slides by incubation with 100 to 300 pL of TNB buffer. This may be performed for 30 min in the humidified chamber of the cyler with biotin-labeled probes 100 to 300 pL of SA-HRP (streptavidin-horseradish peroxidase from the TSA kit) diluted 1 100 in TNB buffer....

Primed In Situ Hybridization

Programmable thermal cycler equipped with a flat plate for holding slides (MISHA, Shandon Lipshaw, Pittsburgh, PA see Note 1). 2. Dimethylsulfoxide (DMSO Sigma Genosys, St. Louis, MO) molecular biology or high-performance liquid chromatography-grade. 3. Ethanol (Fischer Scientific). 4. Oligonucleotide primers (Research Genetics, Huntsville AL see Note 2). The following probes for the SRY gene were high-performance liquid chromatogra-phy-purified, and stored at -20 C 5'-GCAGGGCAAGTAGTCAACGTT-3'...

Kada Krabchi Macoura Gadji Ju Yan and Regen Drouin

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...

Development and Adaptation of the Prins Technology

The advent of molecular genetic techniques has brought forth new procedures for in situ chromosomal analysis. One of these techniques is the primed in situ labeling (PRINS) procedure, which constitutes a fast and efficient alternative to conventional fluorescence in situ hybridization for nucleic acid detection. Based on the use of chromosome-specific primers, the PRINS method combines the high sensitivity of the PCR reaction with the cytological localization of DNA sequences. Since its...

Quantitative PRINS

With the PRINS design, high concentrations of small probes that penetrate the specimen well can be used, making targets effectively saturated and the reaction quantitative. This is particularly true for the dideoxy-PRINS, which is used for measuring the size of telomeric repeat domains at individual chromosome ends, as well as for sizing of other simple repeat domains that vary in size, such as trinucleotide repeats. Evaluation of quantitative PRINS reactions can be done in either of two ways....

Initiation of Lymphocyte Culture

Two cultures are initiated for each subject. 2. For each culture, add 10 mL of culture medium to two 15-mL centrifuge tubes. 3. Using a Pasteur pipet, inoculate each centrifuge tube with 12 to 15 drops of whole blood. Each centrifuge tube represents a separate lymphocyte culture. Label the tubes. 4. Add 0.15 mL of PHA to each tube and mix well. 5. Incubate the cultures at 37 C for 72 h (in general, the cultures may remain undisturbed for the full 3-d period, although some groups gently agitate...

In Situ PCR for the Detection of Human Cytomegalovirus in Suspension Cells During the Latent Phase of Infection

Cytomegalovirus latency depends on an interaction with hematopoietic cells in bone marrow and peripheral blood. The distribution of latent viral DNA and transcripts in these cells was investigated using methods based on polymerase chain reaction (PCR)-driven in situ hybridization (ISH) and reverse transcription (RT)-PCR-driven ISH. Using a conventional thermal cycler, latent viral DNA or transcripts were amplified within suspension cells. Amplified products were then detected by nonisotopic ISH...

Harvesting the Lymphocytes

Using an 18-gage syringe needle, add five drops of reconstituted colcemid (10 pg mL) to each culture. 2. After mixing well, incubate the cultures for 1-2 h at 37 C. 3. Centrifuge at 250 for 10 min. 4. Remove the supernatant and break up the pellet by agitation with a vortex mixer. 5. Add 10 mL of hypotonic KCl-Na citrate mixture to each culture. Suspend the cells by gently inverting the centrifuge tubes manually. Allow the tubes to stand at room temperature for 30 min. 6. Gently suspend the...

Info

RT-PCR mis million uilhoul labelled nucleotides or primer IN-SITU HYBRIDIZATION WITH LABELLED PROBE 1 v il linked primnr '. i IU -( h Fig. 1. Schema of in situ RT-PCR. Direct in situ RT-PCR-labeled nucleotide or primer is incorporated in RT-PCR mix reaction. Indirect in situ RT-PCR-labeled probe is incorporated during a hybridization step. As with all RNA work, precautions against RNase contamination should be taken. All solutions were prepared using 0 1 diethylpyrocarbonate-(DEPC) treated...

Materials

Slide frames or self-sealing solution. 3. Coplin jars and glass staining dishes. 4. 2 Paraformaldehyde add 12 g of paraformaldehyde (Merck ultra pure Art no. 4005) to 600 mL of 1X phosphate-buffered saline (PBS) and heat at 65 C for From Methods in Molecular Biology, vol. 334 PRINS and In Situ PCR Protocols, Second Ed. Edited by F. Pellestor Humana Press Inc., Totowa, NJ 10 min. When the solution starts to clear, add four drops of 10 N NaOH and stir. Adjust...

Chromosome Preparation

Vigorously growing roots of Vicia grandiflora. 2. Digestion mix 2.5 pectolyase Y-23 (MP Biomedicals) and 2.5 cellulase Onozuka R-10 (Yakult Pharmaceutical Industry, Tokyo, Japan) dissolved in 75 mMKCl and 7.5 mMEDTA, pH 4.0. Store the mix in 200-pL aliquots at -20 C. 4. Microscope slides and cover slips (Menzel GmbH., Braunschweig, Germany). 5. Acid-cleaned slides Working in a fume hood, prepare 100 mL of saturated solution of K2Cr2O7 in deionized water and slowly add 50 mL of concentrated...

Observation and Image Acquisition

Classical acquisitions are made using a Zeiss epifluorescent microscope equipped with tri-CCD camera and Vysis computer software (Smart capture 2). An example of simultaneous labeling obtained is illustrated in Fig. 2 with the following specific labeling chromosome centromeres labeled in green by PRINS, achromatic spindle in red by IIF of the P tubulin, and the whole DNA in blue using DAPI as counterstains. 2. Confocal acquisitions Refined images are captured using a Zeiss LMS 510 laser...

Latest Developments in In Situ PCR

Since the first publication on the method of in situ polymerase chain reaction (PCR), several thousand research papers have appeared in peer-reviewed journals describing various findings based solely on the application of this method or combined with other more robust methods, including solution-based PCR, immunohistochemistry, Southern blot, etc. A few years after the advent of PCR, several investigators developed in situ PCR methods that differed considerably from each other with regard to...

Slide Treatments

Place the slides with adhered tissue on a heat-block at 105 C for 5 to 120 s to stabilize the cells or tissues (see Note 6). 1. Place the slides in a solution of 4 paraformaldehyde in PBS for 4 h at room temperature. Using the recommended Coplin jars or staining dishes facilitates these steps. 2. Wash the slides once with 3X PBS for 10 min, agitating periodically with an up and down motion. 3. Wash the slides with 1X PBS for 10 min, agitating periodically with an up and down motion. Repeat...

Franck Pellestor and Petra Paulasova Summary

Both primed in situ labeling PRINS and peptide nucleic acid PNA technologies have emerged as research techniques, but they have quickly evolved to applications in biological diagnosis assays. The two procedures present several features specificity, discriminating ability, rapidity that make them very attractive for cytogenetic purposes. The combined use of PRINS and PNA for in situ chromosomal detection on a same cell preparation is described in this chapter. Key Words PRINS PNA in situ...

Human Sperm Preparation

Phosphate-buffered saline PBS Gibco BRL, Eragny, France . 2. 99 Methanol Prolabo, Paris, France . 3. Ethanol series 70 , 90 , 100 Prolabo . 4. Glacial acetic acid Prolabo . 5. 0.5 MNaOH Merck Eurolab, Nogent-sur-Marne, France . 6. 20X Standard saline citrate SSC 3 MNaCl, 0.3 Mtrisodium citrate, pH 7.5 this solution can be stored for several months at room temperature . 7. Washing buffer 2X SSC diluted from 20X SSC. Fig. 1. Principles of the multicolor PRINS reaction modified from Yan 10J ....

Detection and Microscopy

Antifade solution Vectashield Vector Labs, Burlingame, CA . 4. Cover slips 20 x 40 mm CML . 6. Epifluorescence Microscope Leica DMRB Leica, France equipped with x40 and x100 Plan FluoTar objectives and with a DAPI single band-pass filter Leitz filter A, cat. no. 513804 , an FITC single band-pass filter filter I3, cat. no. 513808 , a tetramethylrhodamine isothiocyanate TRITC single band-pass filter filter N2.1, cat. no. 513812 , an FITC TRITC double band-pass filter...

M E T H O D S I N M O L E C U L A R B I O L O G Y

Metabolomics Methods and Protocols, edited by Wolfram Weckworth, 2007 357. Cardiovascular Proteomics Methods and Protocols, edited by Fernando Vivanco, 2006 356. Cellomics Methods and Protocols, edited by Ken Guiliano, D. Lansing Taylor, and Jeffrey Haskins, 2006 355. Plant Proteomics Methods and Protocols, edited by Herv Thiellement, Michel Zivy, Catherine Damerval, and Valerie Mechin, 2006 354. Plant-Pathogen Interactions Methods and Protocols, edited by Pamela C. Ronald, 2006 353. DNA...

Microdissection

It should be noted that we describe only micromanipulator-based microdissection. Laser capture microdissection, a powerful technique, is more often used in tissue isolation, and is less frequently used to isolate such small-mass items as chromosomes. 1. Inverted microscope Zeiss Axiovert an upright microscope is unsuitable because the glass needle will not fit between the objective and the cover slip. It should have phase contrast, and minimum two objectives x10 and x100. It is unnecessary to...