Introduction for Genetics and Oncology Volume
Rapid Cycle Real-Time PCR
Methods and Applications
The variant expression of genes and their protein products is one of the fundamental mechanisms of pathogenesis in human disease. Variant expression of
genes includes both altered levels of normal gene products and modified proteins
resulting from mutations in exons. In either case, detection of variant expression
during early pathogenesis can provide a rational therapeutic approach.
Recently, a new technology has been developed to detect variant gene expression based on the polymerase chain reaction. Desired segments of nucleic acids
are amplified and quantified very quickly in real-time with great accuracy. This
technology combines rapid temperature cycling with fluorescent detection of
products and has become known as, "Rapid Cycle Real-Time PCR". The methods
described in this volume address detection of mutations and mRNA quantification of a variety of target genes. In addition, to assess the stability of the genome
by analysis of microsatellite sequences are presented. Mutation detection methods usually use hybridization probes, whereas for mRNA quantification, both
SYBR Green I and hybridization probes are commonly used.
The LightCycler (Roche Diagnostics) used by the contributors to this volume
was the first system that permitted the investigator to evaluate target gene
amplification after each cycle in real time. Recently, an automated nucleic acid
extraction system, the MagNAPure LC (Roche Diagnostics), was introduced to
complement the LightCycler system. The combination of MagNAPure LC and
LightCycler now permits the complete automation of extraction, purification,
concentration, amplification and detection of target nucleic acid from patient
samples. In addition, immediate melting curve analysis after PCR with hybridization probes or PCR products is extraordinarily useful. In this volume, the
editors Dr. Dietmaier, Professor Wittwer and Dr. Sivasubramanian, have used
this feature to determine the stability of the genome in micro satellite regions.
This innovative use aids in the diagnosis of hereditary non-polyposis colorectal
cancers.
Real time PCR technology also has many applications in human heart failure
research, diagnosis and therapy. For example, current therapeutic treatment of
human heart failure is based mostly on physiological measurements of heart
function. However, physicians and researchers are handicapped because of the
inability to determine the biochemistry of the heart during therapeutic treatment. As a first step to meet this deficiency, Zhu et al. in this volume present a
method to quantify mRNAs in myocardial biopsy samples of 1 mg size. This
method was recently used to understand the biochemical basis of hebernating
.,
Rapid Cycle Real-Time PCR
Methods and Applications
The variant expression of genes and their protein products is one of the fundamental mechanisms of pathogenesis in human disease. Variant expression of
genes includes both altered levels of normal gene products and modified proteins
resulting from mutations in exons. In either case, detection of variant expression
during early pathogenesis can provide a rational therapeutic approach.
Recently, a new technology has been developed to detect variant gene expression based on the polymerase chain reaction. Desired segments of nucleic acids
are amplified and quantified very quickly in real-time with great accuracy. This
technology combines rapid temperature cycling with fluorescent detection of
products and has become known as, "Rapid Cycle Real-Time PCR". The methods
described in this volume address detection of mutations and mRNA quantification of a variety of target genes. In addition, to assess the stability of the genome
by analysis of microsatellite sequences are presented. Mutation detection methods usually use hybridization probes, whereas for mRNA quantification, both
SYBR Green I and hybridization probes are commonly used.
The LightCycler (Roche Diagnostics) used by the contributors to this volume
was the first system that permitted the investigator to evaluate target gene
amplification after each cycle in real time. Recently, an automated nucleic acid
extraction system, the MagNAPure LC (Roche Diagnostics), was introduced to
complement the LightCycler system. The combination of MagNAPure LC and
LightCycler now permits the complete automation of extraction, purification,
concentration, amplification and detection of target nucleic acid from patient
samples. In addition, immediate melting curve analysis after PCR with hybridization probes or PCR products is extraordinarily useful. In this volume, the
editors Dr. Dietmaier, Professor Wittwer and Dr. Sivasubramanian, have used
this feature to determine the stability of the genome in micro satellite regions.
This innovative use aids in the diagnosis of hereditary non-polyposis colorectal
cancers.
Real time PCR technology also has many applications in human heart failure
research, diagnosis and therapy. For example, current therapeutic treatment of
human heart failure is based mostly on physiological measurements of heart
function. However, physicians and researchers are handicapped because of the
inability to determine the biochemistry of the heart during therapeutic treatment. As a first step to meet this deficiency, Zhu et al. in this volume present a
method to quantify mRNAs in myocardial biopsy samples of 1 mg size. This
method was recently used to understand the biochemical basis of hebernating
.,