Techne PrimeQ User Manual download pdf (Page 41)

2.1 Introduction to PrimeQ

2.1.1 Principle of a real-time PCR instrument

A real-time polymerase chain reaction (PCR) system performs three main functions:

•

Cycles the PCR reagents through the specified temperature profile using a specially

designed thermal block.

•

Excites the fluorescent dye at the appropriate wavelength and at the appropriate point(s) in

the PCR program.

•

Detects the emitted fluorescence in each well during the specified read point - this

information is then fed back to the application software for analysis.

2.1.2 Principle of PrimeQ

PrimeQ is a real-time PCR system that combines a thermal cycler, solid state white light excitation

source and a highly sensitive fluorescence detection system all within the convenience of a

standard 96-well format. Combined with Quansoft, the system’s powerful analysis application

software, real-time quantification and analysis is made fast, easy and accurate. PrimeQ has been

designed with the advantage of an open chemistry format that gives the user full flexibility in the

methods and research they wish to pursue. The system is able to detect as little as 1.0nM

fluorescein and achieve a dynamic range of at least nine orders of magnitude†.

In line with the real-time principle, PrimeQ measures the amplification of DNA via a proportional

increase in fluorescence which is fed back to the application software. This allows the software to

calculate the starting DNA concentration by comparison to a set of standards. The system is

capable of measuring up to four different fluorophores per well in real time, thus opening up the

possibility of multiplex experiments, where multiple dyes are conveniently used within the same

reaction.

The time taken for PrimeQ to read a plate can be adjusted by varying the integration time. Read

times can be programmed from 250ms/well integration time (30 seconds/ 96-well plate) down to

just 50ms/well (10 seconds/plate) for brighter fluorophores. The default integration time is

150ms/well, which gives a read time of around 20 seconds for a 96 well plate.

2.1.3 Applications of PrimeQ

Quantitative real-time PCR:

Monitoring the accumulation of a PCR product as the run

progresses by detecting the increase in fluorescence. Data collection in the early exponential

phase of the PCR allows the Quansoft software to accurately calculate initial template

quantities.

Qualitative (end-point) PCR:

At the end of a run, the instrument is programmed to read the

plate a user-defined number of times for as many fluorophores as present in the samples. The

result indicates positive or negative amplification and the data can be analysed in a number of

ways to give a qualitative result.

Dissociation point determination:

Samples are slowly heated from the annealing

temperature (typically 50 to 65°C) up to the denaturation temperature (~95°C) in small steps.

Fluorescence is measured at each step to determine the amount of dye bound to the dsDNA.

The point at which the two strands separate is associated with a decrease in the fluorescence

signal as the dye can no longer intercalate with the two strands. It is therefore possible to

determine the temperature at which 50% of the double-stranded DNA is dissociated (Tm).

Since the dissociation temperature of a DNA product is characteristic of the GC content, length

and sequence, the Tm can be a useful tool in product identification.

† The dynamic range is assay dependent.

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