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Single-Copy and Point Mutation Analysis (Rolling-Circle PRINS)

  • Jørn Koch
Part of the Methods in Molecular Biology™ book series (MIMB, volume 334)

Abstract

This protocol is a prototype for a series of upcoming procedures aimed at the detection of single molecules of nucleic acids in situ. It uses circular probes that, upon hybridization to their target molecule, can template rolling-circle DNA synthesis, if the target also provides a primer for initiation of the DNA synthesis. This primer may be endogenous or artificial, for example, obtained through cleavage with a restriction enzyme. In this format, the primed in situ labeling (PRINS) product takes the form of a long-tandem repeat copy of the probe, covalently attached to the site of synthesis, and tagged it with copies of the probe. The amplification is sufficient for the detection of single, oligonucleotide size, targets and, depending on the probe format, the probe may detect variations in the targets down to single base substitutions.

Key Words

PRINS RCA rolling circle 

Notes

Acknowledgment

The joint work to develop this protocol was supported by an exchange grant from Uppsala University.

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Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Jørn Koch
    • 1
  1. 1.Institute of Pathology, Aarhus SygehusAarhus UniversityDenmark

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