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 amplify a DNA library made from a single template. For this purpose, preamplifying the template before microdissection can yield excellent results.

In situ applications of polymerase chain reaction (PCR) are used primarily to detect targets too small to be seen with traditional in situ hybridization techniques (4). In certain cases, they also can provide increased specificity and more rapid analyses. In addition to target location, however, PCR in situ and other genome amplification techniques can be used to increase the total amount of DNA associated with a genome or with a specific region of a genome (5,6).

From: Methods in Molecular Biology, vol. 334: PRINS and In Situ PCR Protocols, Second Ed.

Edited by: F. Pellestor © Humana Press Inc., Totowa, NJ

The techniques described in this chapter can be used in either eukaryotic or prokaryotic cells and are described to enable results directly from following the protocols but can be generalized to allow them to be adapted to specific requirements.

2. Materials

2.1. Slide Preparation

1. Fixative: 3:1 methanol:glacial acetic acid.

2. Methanol 100%.

3. 0.075 KCl hypotonic solution.

4. Centrifuge.

5. Falcon tube.

6. Microscope slide.

2.2. Degenerative Oligonucleotide-Primed PCR In Situ

1. Template cover slip.

2. Thermo Sequenase DNA Polymerase, Thermo Sequenase reaction buffer (Amersham, Arlington Heights, IL). Aliquot upon arrival and store at -20°C until needed (see Note 1).

3. dNTP mix (Roche, Indianapolis, IN): Dilute each dNTP dATP, dCTP, dGTP, and dTTP, each at a stock concentration of 100 mM) in dH2O to a working concentration of 2 mM per dNTP. Aliquot and store at -20°C.

4. Tetramethylrhodamine-6-dUTP, stock 1 mM (Molecular Probes, Eugene, OR). Dilute to 40 pM and aliquot. Store at -20°C (see Note 2).

5. Degenerative oligonucleotide-primed primer (5'CCGACTCGAGNNNNNN ATGTGG-3'). Resuspend with 1X Tris-EDTA buffer, pH 8.0, to a stock concentration of 40 pM Aliquot and store at -20°C.

6. 20X Standard saline citrate (SSC): Thoroughly dissolve 174.3 g of NaCl and 88.2 g of trisodium citrate in 800 mL of double distilled H2O. Bring to a final volume of 1 L with ddH2O and adjust the pH to 5.3 with concentrated HCl. Adjusting the pH of the 20X stock to 5.3 will produce a pH of 7.25 when diluted to a 2X SSC/70% formamide solution. Store at room temperature until needed.

7. 4X SSC, 0.1% Triton X-100: Dilute 20X SSC to a final concentration of 4X SSC, 0.1% Triton X-100 (by volume) by adding the appropriate amount of Triton X-100 and diluting with dH2O. Store at room temperature.

8. Mounting medium: 0.3 pg/mL Vectashield with DAPI (Vector Laboratories Inc., Burlingame, CA).

9. Rubber cement. Store at room temperature.

10. Clean microscope slides.

11. Clean tissues.

12. Zeiss Axioskop (Carl Zeiss, Inc, Thornwood, NY) and images captured by a Vysis QUIPS Imaging Analysis System (Vysis, Downers Grove, IL).

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