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Real Time RT-PCR

Real Time RT-PCR

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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Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

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LightCycler technology in molecular diagnostics.

Elaine Lyon¹, Carl T Wittwer

¹Department of Pathology, University of Utah, Salt Lake City, Utah, USA. lyone@aruplab.com

The Journal of Molecular Diagnostics : JMD

|February 7, 2009

Summary

This summary is machine-generated.

LightCycler technology offers rapid, quantitative polymerase chain reaction (PCR) with real-time monitoring and melting curve analysis for diverse molecular pathology applications. This review highlights 15 years of advancements in clinical laboratory use.

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Area of Science:

Molecular Biology
Clinical Pathology

Background:

LightCycler technology integrates rapid-cycle PCR with real-time fluorescence and melting curve analysis.
Established in 1997, it has become a key tool in molecular pathology for oncology, inherited diseases, and infectious diseases.

Discussion:

Quantitative PCR is achieved in 15-30 minutes using a closed-tube system by monitoring product accumulation.
Melting curve analysis enables genotyping and haplotyping, with novel mutations detected by altered melting temperatures or curve shapes.
Various melting probe designs, including adjacent hybridization probes, single labeled probes, unlabeled probes, and snapback primers, are utilized.

Key Insights:

High-resolution melting facilitates mutation scanning by identifying heterozygous changes.
The technology provides rapid and accurate molecular diagnostics in clinical settings.
Advances over 15 years have expanded its utility in various diagnostic areas.

Outlook:

Continued development of LightCycler technology promises further improvements in diagnostic speed and accuracy.
Potential for broader application in personalized medicine and complex genetic analyses.
Integration with other high-throughput technologies for comprehensive genomic profiling.