PCR reaction mixture for fluorescence-based gene expression...

Details PCR reaction mixture for fluorescence-based gene expression... PCR reaction mixture for fluorescence-based gene expression...

2001-01-03

2003-05-06

Riley, Jezia (Department: 1656)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091200

Reexamination Certificate

active

06558929

ABSTRACT:

BACKGROUND OF THE INVENTION
Gene expression occupies a key function in the evaluation of molecular processes in the body and great efforts are being made to investigate the significance of the expression of numerous genes also as a result of drug effects and to check it with respect to its predictive value in regard to the course of an illness and the success of a therapy.
A prerequisite for converting genetic information initially is the transcribing of the corresponding DNA sequence into mRNA. Gene expression can be regulated at the level of transcription as well as post-transcriptionally. For evidence-based medicine, the clarification of the mechanisms, which lead to a changed gene expression in the course of an illness, is an important objective, because new therapy concepts can be derived from it, which lead to an improved treatment of patients.
According to estimates of the organizers, the human genome project presumably will come to a conclusion in the year 2001. At the end of this project, which is being conducted worldwide, approximately 140,000 genes will have been identified. The analysis of the gene expression in different cell types and tissues, which provides important information concerning the normal state and the genesis of the pathologic state of cells and tissues, represents the greatest challenge at the present time as well as in the post-genome epoch.
A plurality of methods is employed for the analysis of gene expression, including Northern Blot and RT-PCR techniques. By means of chip-based technologies, that is, planar carriers of plastic, glass, gelatin, etc., on the surface of which a plurality of different (DNA) molecules, the positions of which are known and can be addressed, are disposed, thousands of genes can be investigated simultaneously with respect to their expression.
Aside from the different methods of analyzing gene expression, new technologies are being developed, in order to be able to detect nucleotide polymorphisms (that is, different variations of a gene) systematically, and to be able to evaluate them with respect to their biological significance in the course of an illness and, in the case of a medical application, in the sense of an individualized therapy.
A relatively recent method for fluorescence-based gene expression analyses and gene mutation analyses is represented by the investigation of amplified probes by the PCR technique in real-time PCR analytical equipment, such as the Lightcycler (Roche Diagnostics), TaqMan (Perkin-Elmer), etc.
The LightCycler is equipped with a three-channel fluorimeter, which can detect fluorescence at 530 nm (SYBR-Green), 640 nm (LC-RED 640) and 705 nm (LC-RED 7050). The manufacturer, Roche Diagnostics makes several tests available for the PCR amplification in the LightCycler, which can be used depending on the type of labeling (SYBR-Green or FRET methods; see below).
Dye Labeling
In order to be able to measure the newly synthesized in DNA in the subsequent real-time PCR, the DNA must be labeled by suitable dyes, which can be detected. For detecting the fluorescence signals, various real-time PCR detection systems were developed, with which it is possible to follow the whole of the PCR reaction. In the following, basic principles of the detection are explained:
1.) Fluorescence resonance energy transfer (FRET) is a process, for which a donor molecule, fluorescing after being stimulated by short-wave light, transfers its emission energy to a second acceptor molecule, which reacts to this with the emission of light of longer wavelength. The energy transfer from the one to the other molecule takes place over electron flow. The reporter molecule provides information concerning the product increase during the PCR. The so-called quencher molecule absorbs the fluorescence signals of the reporter molecule as long as both molecules are directly adjacent to one another in the hybridization probe. In this basic state, the reporter emission radiation for the fluorescence detector, with which the product increase in the PCR is measured, is invisible. Only as the PCR product increases, is there a spatial separation from the reporter and the quencher molecule. By these means, the reporter fluorescence becomes detectable and correlates directly with the amount of PCR product formed in the reaction.
A further method is referred to as the TaqMan method. In the case of the TaqMan method (or 5′-nuclease assay), the fluorescence-labeled hybridization probes bond to the complementary target strand between the primer binding sites. For the synthesis of the new strand, the hybridization probe is cut into small fragments by the 5′-3′-exonuclease activity of the Taq polymerases and released from the target strand. The reporter molecules and the quencher molecules are now present separately in the reaction mixture and the measured increase in the reporter fluorescence per PCR cycle correlates directly with the increase in the PCR product.
Other hybridization probes, synthesized according to the FRET principle, can be used for carrying out mutation analyses. Particularly important is the detection of so-called single nucleotide polymorphisms (SNPs), which come about due to the exchange of individual bases. Two hybridization probes, each labeled with a fluorescence dye, are used for the SNP analysis.
One donor probe binds directly adjacent to the mutation region. A second probe is produced so that it binds either complimentarily to the wild type or over the mutation site. In the melting point analysis, carried out after the PCR, the probe melts off at a particular temperature. If the probe bonds complimentarily to the wild type, it melts off at higher temperatures. On the other hand, in the presence of a mutation, the probe melts at lower temperatures. A mutation analysis therefore becomes possible. The fluorescence decline is calculated as a negative first derivative (as a melting peak). The mutation can be diagnosed by the displaced melting curve. This method with different fluorescence dyes can only be used by means of a real time detection system.
The principle of the real time detection system also forms the basis of the LightCycler from Roche Diagnostics. The LightCycler has three channels by means of which the emitted light quanta of dyes can be detected. DNA can by labeled with SYBR-Green; in addition, FRET probes, which are labeled with LC-Red 640 or LC-Red 705 dyes can also be used.
If the FRET method is used in the LightCycler, special hybridization probes must be added to the reaction mixture. They are labeled with fluorescein and LC red 640 or LC red 705 (from Roche Diagnostics). The fluorescence is observed only if both probes (donor probe and acceptor probe) have bonded in the immediate spatial vicinity to the target sequence. The transfer of light quanta (h*v), namely the fluorescence resonance energy transfer (FRET; see FIG.
1
), then comes about.
2.) In the SYBR-Green method, the SYBR-Green intercalates in each case between two complementary base strands during the DNA synthesis and, with that, experiences a measurable increase in fluorescence as the PCR reaction progresses (see FIG.
2
). However, the use of SYBR-Green lacks any specificity with regard to the template, which is to be investigated (that is, the DNA binding site), because the primer dimers, which are formed during the reaction, also cause an increase in fluorescence. Initially, this cannot be differentiated from the desired DNA synthesis product and can lead to wrong interpretations. However, it is possible to differentiate between the specific product and primer dimers at the end of the PCR by means of a melting curve analysis. For this, the PCR products are heated continuously over a particular temperature range and are present only as a single strand, depending on their melting point. The decrease in fluorescence, associated with this, is recorded. Smaller fragments, such as the primer dimers, have a melting point, which is lower than that of larger PCR products.
The representation of the fluorescence signal changes as a f

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