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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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Related Experiment Video

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Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis
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RAPID LOW-COST DETECTION OF TYPE 2CALR MUTATION BY ALLELE-SPECIFIC RT-PCR FOR DIAGNOSIS OF MYELOPROLIFERATIVE

M V Dybkov1, M P Zavelevich2, D F Gluzman2

  • 1Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv 03143, Ukraine.

Experimental Oncology
|May 13, 2022
PubMed
Summary

This study introduces a rapid, low-cost allele-specific RT-PCR method for detecting CALR type 2 mutations in myeloproliferative neoplasms (MPNs). This technique offers a cost-effective alternative for diagnosing essential thrombocythemia (ET) and primary myelofibrosis.

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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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Area of Science:

  • Hematology
  • Molecular Biology
  • Genetics

Background:

  • Calreticulin (CALR) gene mutations are found in 15-24% of essential thrombocythemia (ET) and 25-35% of primary myelofibrosis (PMF) cases.
  • Current methods for detecting CALR mutations, such as Sanger sequencing and next-generation sequencing, are expensive and require specialized equipment.
  • Type 1 CALR mutations can be detected using polymerase chain reaction (PCR) and agarose gel electrophoresis.

Purpose of the Study:

  • To develop and validate an allele-specific reverse transcription (RT-PCR) assay for rapid, low-cost detection of the type 2 CALR mutation.
  • To provide a more accessible diagnostic tool for CALR-mutated myeloproliferative neoplasms (MPNs).

Main Methods:

  • Allele-specific primers were designed to detect the type 2 CALR mutation (5-bp insertion; c.1154_1155 ins TTGTC).
  • The assay was tested on cDNA from a patient with JAK2- and MPL-negative ET who had a confirmed CALR mutation via Sanger sequencing.
  • RT-PCR products were analyzed using agarose gel electrophoresis.

Main Results:

  • The allele-specific RT-PCR successfully detected the type 2 CALR mutation (K385fs*47 ins5) in the patient sample.
  • Results from the RT-PCR assay were consistent with those obtained from Sanger sequencing.
  • Agarose gel electrophoresis clearly distinguished between normal and type 2 mutated CALR alleles.

Conclusions:

  • Allele-specific RT-PCR provides a fast and low-cost method for detecting the common type 2 CALR mutation (ins 5) in MPN patients.
  • This assay can serve as a valuable tool for routine diagnostics in clinical settings.
  • The method offers an affordable alternative for identifying CALR mutations, improving accessibility to molecular diagnostics.