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In vitro Mutagenesis

In vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

In-vitro Mutagenesis

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

Spontaneous and Induced Mutations

Spontaneous and Induced Mutations

Spontaneous mutations arise infrequently during DNA replication due to errors in the process. A key factor behind these errors is tautomeric shifts in nitrogenous bases, where bases transition from keto to enol forms or amino to imino forms. This shift can alter base-pairing rules, leading to mutations. Additionally, reactive oxygen species (ROS) arising from aerobic metabolism can damage DNA, resulting in depurination (loss of a purine base) or depyrimidination (loss of a pyrimidine base).

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Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation

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Published on: August 23, 2024

The Pyrosequencing-Based Method for JAK2 Exon 12 Somatic Mutation Detection.

Elena Pozdysheva¹, Tatiana Subbotina^2,3, Yana Voytsekhovskaya¹

¹Central Research Institute of Epidemiology Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing of Russian Federation, 111123 Moscow, Russia.

Diagnostics (Basel, Switzerland)

|June 26, 2026

Summary

This summary is machine-generated.

A new pyrosequencing method accurately detects JAK2 exon 12 mutations, crucial for diagnosing myeloproliferative neoplasms (MPN) and polycythemia vera (PV). While effective, complex mutations may require Sanger sequencing confirmation.

Keywords:

JAK2 exon 12 myeloproliferative disease polycythemia vera pyrosequencing

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A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

Published on: December 7, 2014

Area of Science:

Molecular Biology
Oncology
Genetics

Background:

JAK2 exon 12 mutations are key diagnostic markers for myeloproliferative neoplasms (MPN).
These mutations are integral to the World Health Organization's diagnostic criteria for polycythemia vera (PV).
Accurate detection is vital for differential diagnosis in MPN cases.

Purpose of the Study:

To develop and validate a pyrosequencing technique for detecting somatic mutations in JAK2 exon 12.
To assess the qualitative and quantitative performance of this pyrosequencing method.
To evaluate the method's utility in a cohort of polycythemia vera patients.

Main Methods:

PCR and pyrosequencing primers were designed for the JAK2 exon 12 region.
PCR conditions were optimized for pyrosequencing-based detection.
Analytical sensitivity and specificity were determined using plasmid controls, and the method was validated on 145 MPN patient DNA samples.

Main Results:

The pyrosequencing method demonstrated analytical sensitivity with a limit of detection ranging from 3.4% to 9.9%.
JAK2 exon 12 mutations were identified in 4.8% (7 of 145) of MPN patients.
Complex mutations required Sanger sequencing confirmation, and allele burden changes were observed over time in one patient.

Conclusions:

The developed pyrosequencing method offers a straightforward approach for detecting JAK2 exon 12 mutations.
The technique provides both qualitative and quantitative analysis capabilities.
Manual interpretation and Sanger confirmation may be necessary for certain complex JAK2 exon 12 mutation types.