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Related Experiment Video

Updated: Jul 10, 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

Microarray-based DNA resequencing using 3' blocked primers.

Jakub Sram¹, Steve S Sommer, Qiang Liu

¹Department of Molecular Genetics, City of Hope National Medical Center, Duarte, CA 91010, USA.

Analytical Biochemistry

|November 21, 2007

Summary

This summary is machine-generated.

Pyrophosphorolysis-activated polymerization (PAP) with blocked primers offers ultra-high specificity for nucleic acid amplification. This novel sequencing method accurately identifies genetic variations, including single-base substitutions and mutations, with high throughput potential.

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3' End Sequencing Library Preparation with A-seq2

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3' End Sequencing Library Preparation with A-seq2

3' End Sequencing Library Preparation with A-seq2

Published on: October 10, 2017

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Conventional sequencing technologies face limitations in throughput and accuracy.
Nucleic acid amplification is crucial for various genomic applications.
The need for highly specific and efficient sequencing methods is paramount.

Purpose of the Study:

To evaluate pyrophosphorolysis-activated polymerization (PAP) as a novel nucleic acid amplification method.
To assess the specificity and accuracy of PAP using 3' blocked primers (P*s) for resequencing applications.
To demonstrate the potential of PAP for high-throughput microarray-based resequencing.

Main Methods:

Developed and tested pyrophosphorolysis-activated polymerization (PAP) utilizing 3' blocked primers (P*s).
Applied PAP to resequence a 20-bp region of the factor IX gene using a microarray.
Analyzed the specificity of P*s in discriminating 3' end mismatches and internal mismatches.

Main Results:

P*s demonstrated ultra-high specificity for 3' end mismatches and high specificity for internal mismatches.
Successfully identified two wild-type samples and all introduced genetic variations, including single-base substitutions, microdeletion, microinsertion, and heterozygous mutation.
Achieved signal/noise ratios exceeding 1000 between matched and mismatched P*s, despite primer purity limitations.

Conclusions:

PAP resequencing with P*s exhibits exceptional specificity and accuracy for genetic variation detection.
The method shows significant potential for accurate, high-throughput, microarray-based resequencing.
PAP represents a promising advancement in nucleic acid sequencing technologies.