LMPC of Living Cells
Common laser microdissection and capture techniques require a dry environment and are therefore restricted to fixed or frozen tissue sections or cell preparations (30,33). In contrast, living cells need medium for growth and survival. Beyond the necessity of a humid environment, contamination is especially critical with living specimens, if the captured cells will be reculti-vated after microdissection. Therefore an entirely noncontact microdissection and capture method is needed for isolation...
Coupling of Amino Modified Oligonucleotide Capture Probes to Carboxylated Microspheres
Microspheres should be protected from prolonged exposure to light throughout this procedure. 1. Bring a fresh aliquot of -20 C, desiccated Pierce EDC powder to room temperature (see Note 2). 2. Resuspend the amine-substituted oligonucleotide capture probe to 1 mM in dH2O (see Note 1) 3. Resuspend the stock microspheres by vortex and sonication for approx 20 s. 4. Transfer 5.0 X 106 of the stock microspheres to a USA Scientific microcentrifuge tube (see Note 3). 5. Pellet the stock microspheres...
Glass Mounted Specimen vs Membrane Mounted Specimen
Different sample sources can be used with LMPC. Numerous protocols for sample preparation and downstream application techniques have been published during the past few years for laser micromanipulation and microdissection (1-3,5,15,16,33,40,42-44). Specimens can be prepared either directly onto routine glass slides or on special membrane-spanned slides (See Note 2). Depending on the nature of the sample and the purpose of the experiment, catapulting may be performed directly without prior laser...
Data Interpretation
The ratios calculated above are used to determine whether the run is acceptable. For this to be the case, the three controls that represent the three potential genotypes must yield ratios within the ranges shown in Table 1. In the sample data given in Table 3, MTHFR 677 is the target with the major allele (677C) detected by the 485-nm filter and the minor allele (677T) detected by the 560-nm filter. First we confirm that the NTC signal is between 150 and 200. Then we divide the control values...
Invader Procedure
Take reagents out of the refrigerator, and allow 30 min to come to room temperature. 2. Use the Microsoft Excel worksheet with macros supplied by Third Wave technologies. This quickly adjusts the volumes of reagents based on the number of samples to be tested. 3. Since the reaction is done in a thermal cycler (although a well-calibrated digital dry block would be sufficient) and read in a plate reader, the format is based on the 96-well plate, with four controls. 4. We suggest including at...
PCR Primer Set Stabilizer and Probe Design
The SNP assay requires four components the amplicon, the stabilizer oligo, and the two reporters, each specific for either the wild-type or mutant allele (Fig. 1). The detection method is based on hybridization of a biotinylated single-stranded amplified DNA molecule to the probe mixture. Reporters are designed to be complementary at their 3' end to the polymorphic or mutated target DNA nucleotide and are labeled at their 5' end with a Cy3 Cy5 fluorophore (see Note 1). The stabilizer oligo is...
Technical Setup of the PALM Micro Beam System
A fully robotic unit called RoboMover functions as a multipurpose collection device, with adapters for routine microfuge tubes, multicap strips, and microtiter plate formats. Guided by the entries in the element list (Fig. 5 see Color Plate 4 following p.18.), complex experimental setups can be planned and processed automatically. The highly automated sample capture device is supported by the second generation of RoboStage. This newly developed microscope stage can travel large distances in x y...
Data Analysis and Genotype Assignment
A quantification program such as the one supplied with ScanArray Express handles the scanning images and quantitates the signals from each spot. The raw data are collected in an Excel sheet. 1. Subtract the background, measured either around the spots or at negative control spots, i.e., spotted cTags without corresponding tagged primers, from the signals measured in each channel. 2. Assign the genotypes of the SNPs in each sample by calculating the ratios between the signals from one of the...
Multiplex Nested RTPCR
Sequence data for SARS CoV were obtained from the curated database in GenBank. The unique and conserved regions of SARS CoV were selected by aligning the released SARS CoV sequences and the latest nonredundant nucleic acid sequences in the NCBI database (ftp ftp.ncbi.nih.gov). 2. To allow detection of SARS CoV, multiple regions from the open reading frame (ORF) of replicase 1a, spike glycoprotein, and nucleocapsid protein were selected as the targets for hybridization detection. 3. To amplify...
Notes
We recommend the software program Oligo for LDR primer design. This program is also useful in designing PCR and multiplex PCR primers. This program calculates Tm using the nearest neighbor method. Gene-specific PCR and LDR primers are generally designed with Tms around 70 C. To perform multiplex PCR PCR, gene-specific oligonucleotide primers with universal primer sequence attached to the 5' ends are required. The sequence of the universal primer is 5'-ggagcacgctatc-ccgttagac-3'. LDR...
Variations on a Theme
In addition to the standard PCR LDR technique outlined in Subheading 3.2. above, there are several variations of LDR as an assay tool for mutation identification, SNP detection and DNA methylation analysis. To genotype hundreds of thousands SNPs accurately in multiple samples in a high-throughput format, one variation is to perform LDR on the DNA samples followed by PCR amplification of the ligation products. By performing LDR directly on the DNA samples using primers bearing universal...
Biological Results
Several different assays have been performed in cooperation with A. Togl, Ch. Gauer, and R. Kirchner (Advalytix) to demonstrate the usefulness and power of the SAW-mediated sample fluid agitation during hybridization processes. Two such experiments are presented here as representatives of a variety of different assays that have proved to result in a higher signal intensity and improved homogeneity compared with conventional methods. 3.4.1. Rat-Specific Oligonocleotide Microarray Hybridization...
Surface Acoustic Waves
SAWs were first described in combination with earthquakes (4). Reduced to a significantly smaller nanoscale, they found their way into friendlier fields SAW devices are widely used for radio frequency (RF) signal processing and filter applications and also play a large part in mobile communication. SAW devices have been around for years in communication circuitry every cell phone has filters using the effect. An electrical signal fed into so-called transducers on the surface of a piezoelectric...
Applications of Pcrldr and the Universal DNA Microarray
When our approaches are combined with PCR, they have been successfully applied to the simultaneous multiplex detection of numerous genetic diseases (see Subheading 1.4.1. below). In our own laboratory, the approach has been validated on hundreds of clinical tumor samples during detection of 19 K- ras and 110 p53 gene mutations in non-microdissected tumors, as well as stool, demonstrating the ability to find mutations despite a large quantity of background normal sequence (5,10,12-14). Our...
Principles of the Universal DNA Microarray
The Universal DNA microarray is a technology platform that provides an alternate strategy in microarray design. It differs from the conventional approaches to microarray technology in that mutation detection and hybridization to the array surface are completely separate events. Since the specificity for the Universal DNA microarray is determined by LDR, it avoids the false negatives and false positives associated with direct DNA hybridization arrays. For high-throughput detection of specific...
Design of PCR Amplification Primers and Multiplexed PCR Reactions
PCR amplification primers were designed to amplify 19 regions of the CFTR gene containing the mutation sites, with the amplicons ranging from 89 to 212 bp in length (Table 3). One primer of each pair was biotinylated at the 5' terminus for labeling the target strand of the amplicon. Using a small target DNA (approx 100-300 bp) minimizes the potential for steric hindrance to affect the CFTR, cystic fibrosis transmembrane conductance regulator gene. a Primer E19D is used for amplification of both...
References
Sachidanandam, R., Weissman, D., Schmidt, S. C., et al. (2001) A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409, 928-933. 2. Venter, J. C., Adams, M. D., Myers, E. W., et al. (2001). The sequence of the human genome. Science 291, 1304-1351. 3. Lander, E. S., Linton, L. M., Birren, B., et al. (2001). Initial sequencing and analysis of the human genome. Nature 409, 860-921. 4. Syvanen, A. C. (2001). Accessing genetic variation genotyping...
Downstream Applications
For all subsequent molecular analyses of LMPC-generated samples, the best results for the recovery of proteins, DNA, or RNA are achieved with freshly prepared cryosectioned specimens. 3.4.1. Isolation of DNA From Captured Samples For DNA preparations from paraffin sections, it is recommended to use a Proteinase K-containing catapult buffer. For cryosections, it is not necessary to perform catapulting of the cells into a buffer with Proteinase K. 3.4.1.1. Capture of the Samples 1. The capture...
Molecular Basis of Polymorphism Detection Using Invader
The Invader assay detects biallelic polymorphisms through the use of allele-specific dye and quencher, dual-labeled probes, termed fluorescence resonance energy transfer FRET cassettes, and Cleavase, a naturally occurring flap endonuclease. Cleavase digestion separates the dye from the quencher when the FRET cassette forms a specific 3D structure with the 5' portion of a polymorphism-specific oligonucleotide, termed the primary probe 5' flap. This 5' flap, which is not complementary to the...
Paolo Fortina md phd
Center for Translational Medicine, Department of Medicine Jefferson Medical College, Philadelphia, PA 2005 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Medicine is a trademark of The Humana Press Inc. All papers,...
