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Updated: Jul 5, 2026

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

Published on: October 8, 2019

Single-nucleotide sequence discrimination in situ using padlock probes.

Mats Nilsson¹, Ulf Landegren, Dan-Oscar Antson

¹Uppsala University, Uppsala, Sweden.

Current Protocols in Human Genetics

|April 23, 2008

Summary

Padlock probes leverage DNA ligase sensitivity to mismatches for precise DNA sequence variant detection in situ. This method enables highly specific identification of target DNA sequences.

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Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

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Published on: March 29, 2017

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

DNA ligases are enzymes crucial for DNA repair and replication, exhibiting high sensitivity to mismatches at ligation junctions.
This enzymatic property forms the basis for developing sophisticated molecular tools for genetic analysis.
Padlock probes are specifically designed oligonucleotides that exploit this sensitivity for sequence discrimination.

Purpose of the Study:

To present a detailed protocol for distinguishing closely similar DNA sequences in situ using padlock probes.
To explore the application of DNA ligase's mismatch sensitivity for high-specificity DNA detection.
To discuss methods for signal amplification of ligated padlock probes.

Main Methods:

Utilizing padlock probes, which are linear oligonucleotides with target-complementary ends and an internal segment.
Hybridizing padlock probes to target DNA sequences, bringing probe ends into proximity.
Employing DNA ligase to join probe ends only when perfectly matched to the target DNA sequence.
Implementing signal amplification strategies for enhanced detection of circularized probes.

Main Results:

Padlock probes demonstrate high specificity in detecting target DNA sequences due to the requirement for perfect end-matching for ligation.
The protocol allows for in situ discrimination between closely related DNA sequence variants.
Signal amplification methods significantly enhance the detectability of successful probe circularization.

Conclusions:

Padlock probes offer a powerful and specific method for in situ DNA sequence variant discrimination.
The sensitivity of DNA ligases to mismatches is a key feature enabling this precise detection.
This approach, combined with signal amplification, holds significant potential for various genetic analysis applications.