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Cycling Primed In Situ Amplification 425
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Cycling Primed In Situ Amplification
John H. Bull and Lynn Paskins
1. Introduction
Primed in situ amplification (PRINS) is a technique for the visualization of specific
sequences, usually repeat sequences, in fixed cell nuclei. When viewed on the microscope,
the resulting signals can be seen as spots within nuclei, providing a means to
visualize telomeres, centromeric regions, Alu repeats, or other sequences.
At its simplest, the PRINS reaction is a primer extension conducted under a sealed
coverslip on a microscope slide. A mix of oligonucleotide primer, dNTPs (including
a labeled dNTP), and Taq DNA polymerase is applied to a preparation of fixed cell
nuclei on the slide, which is placed on a heating block and subjected to a round of
denaturation, annealing, and extension. During this process, the nuclei are held in place
while the DNA strands are made available for oligonucleotide annealing and extension
by the polymerase. Unincorporated nucleotides are washed off, and the incorporated
labeled nucleotide is detected typically by fluorescence (1,2).
PRINS has many applications in common with the widely used fluorescence in situ
hybridization (FISH) technique (3). However, PRINS has significant advantages in the
speed of the reaction, avoidance of toxic chemicals, lack of dependence on a carefully
controlled stringency wash step, and in the use of easily synthesized oligonucleotides
rather than expensive probes. For example, specific primers can be used for most
human chromosomes or pairs of chromosomes (4), giving discrete subnuclear spots
that allow chromosome enumeration. The target sequence is typically satellite repeat
(e.g., α-satellite, a family of 171-bp repeat units present at the centromeres of human
chromosomes) and the strength of the signal is a function of the number of repeat
units at the target site.
Cycling PRINS represents a modification where the primer extension is repeated
several times (see Fig. 1), with the result that multiple labeled DNA strands are
synthesized with a concomitant increase in signal (5). This has obvious advantages in
sensitivity, but in practice there are constraints to the signal build-up in cycling PRINS,
which center around the fixation method used to prepare the nuclei or cells. PRINS
depends on unimpeded access of reagents to the nuclear DNA. As the newly primed
strand is synthesized, it is held in place by base pairing to the nuclear template DNA.
When the reaction is cycled, there may be nothing to stop the newly synthesized strand
diffusing away from the site of synthesis. In our laboratory, this problem appears
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
425