TaqMan® Technology is the gold standard in gene expression research for performance, quality and content.
Genotyping of RT-PCR using TaqMan probes is a method that requires special compound oligonucleotides – TaqMan probes, conjugated with two ends with fluorescent dyes: a signal fluorophore at the 5 ‘end and a quenching fluorophore at the 3’ end.
TaqMan series. This series includes more than 8 million kits for the analysis of gene expression, genotyping of point polymorphisms (including in relation to different pathologies or metabolic features), analysis of microRNA and gene copy number by real-time PCR.
SYBR® Green I Dye technology
SYBR Green I is a double-stranded DNA-specific intercalating dye. The solution is optimized for use in real-time PCR (RT-PCR). It is recommended to use the dye as a 50X solution; when optimizing the PCR conditions, it can be used in the range from 25X to 100X. The supplied 100% DMSO solution can be added up to a final content of 5%.
Ingredients: SYBR Green I in buffered solution, pH 8.0
Spectral properties: excitation wavelength – 497 nm, fluorescence – 521 nm
How it works: SYBR Green I is an intercalating dye, the fluorescence intensity of which increases by several orders of magnitude when inserted into double-stranded DNA. Therefore, during PCR, with the accumulation of the amplification product, the fluorescence signal increases, forming a sigmoid curve.
Since SYBR Green I binds to any double-stranded DNA, an increase in fluorescence in PCR can be associated with the accumulation of both a specific product and a nonspecific one (primer dimers, nonspecific annealing of primers). In a reaction without a template, the fluorescence level may exceed the threshold level not due to contamination with the template, but due to the formation of primer dimers. To obtain correct results after the end of the reaction, it is necessary to additionally analyze the PCR result by constructing the so-called “melt curve”.
Melting curve analysis
The melting curve should be constructed from 41-50° F below the primer’s melting point (Tm) to full denaturation (approximately 203° F). Typically, the curve is built after the PCR. It is most convenient to analyze the curve in the coordinates -d (RFU) / dT versus t ° (inverse dependence of the velocity on temperature, Fig. B).
SYBR Green I is compatible with any real-time amplification device.
Compatibility with PCR components
50X SYBR Green I is compatible with the reference ROX dye, with any unstained reaction buffers, as well as with any type of polymerases, regardless of the presence of 5 ‘or 3’ exonuclease activity. 50X SYBR Green I can be used in reactions with the addition of dUTP and uracyl glycosylase. When using, it is recommended to use “hot start” polymerases.
The number of freezing / defrosting cycles: no more than 20.
The GenomeLab™ GeXP
Genetic Analysis System
The purpose of genetic analysis is to obtain accurate information, regardless of the source of the research material. The GenomeLab ™ GeXP sequencer provides the flexibility to tackle the difficult task of analyzing inaccurate, inconsistent data. Modern approaches in chemistry are used, as well as unique reagents for DNA sequencing: linear polyacrylamide (LPA) gel, an array of capillaries treated with a special coating, isotachophoresis (ITP) and infrared cyanine dyes. In addition, samples are denatured immediately prior to loading into the capillary array. The result is data that needs less correction and better analysis in general. The capabilities of the GenomeLab ™ GeXP Series enable the sequencing of complex DNA regions where other systems fail.
The GenomeLab ™ GeXP fully automated genetic analysis system builds on Beckman Coulter’s solid expertise in laboratory automation and leadership in capillary electrophoresis systems. GenomeLab ™ GeXP sequencers automatically fill the capillary array with patented linear polyacrylamide (LPA) gel, denature and load samples, run an electrophoresis program, and analyze data. The software will allow you to quickly review the quality of data and automate their assessment, in accordance with the operator’s requirements. Now you can only focus on research. GenomeLab ™ GeXP Sequencers automatically select the optimal sequence to facilitate DNA electrophoresis.
With GenomeLab ™ GeXP instruments, you can perform a complete genetic analysis using one gel, one capillary array, one software.
GenomeLab ™ GeXP Sequencer Capabilities
- Linear Polyacrylamide (LPA) Gel Application: Maximum Performance and Resolution;
- The capillary array is treated with a special coating;
- Measurement of laser-induced fluorescence at four wavelengths;
- 96-well plates are used for samples and buffer;
- Simultaneous reading of eight samples;
- Automatic filling of the capillary array with gel;
- Automatic sample loading and pre-denaturation;
- Easy parameterization for DNA sequencing and fragment analysis;
- The software is designed to work in the Windows XP environment;
- Work with two 96-well plates;
- Built-in barcode reader;
- Advanced data processing software package.
Sequencing complex regions of DNA when other systems fail (de novo)
Modern approaches in chemistry are used, as well as unique reagents for DNA sequencing: linear polyacrylamide (LPA) gel, an array of capillaries treated with a special coating, isotachophoresis (ITP) and infrared cyanine dyes. In addition, samples are denatured immediately prior to loading into the capillary array. The result is data that needs less correction and better analysis in general. GenomeLab ™ GeXP systems are capable of sequencing complex DNA regions where other systems fail.
The instrument fills in the gaps with high-resolution sequences. The figure shows the results of sequencing a complex region of the human genome. The sequence of the site, which could not be determined by another automatic sequencing system, was established using innovative methods applied in the GenomeLab ™ GeXP sequencer.
Some Specifications of the GenomeLab ™ GeXP Sequencer
- using color-coded sequencing run terminators;
- optimized four-color method (DTCS four colors);
- determination of 700 bases in one sample in 100 minutes with an accuracy of more than 98%;
- fragment analysis;
- automatic allele detection;
- Resolution: + 1 base for a fragment length of 400 bases, + 2 for a length of 600 bases.