Techne PrimeQ User Manual download pdf (Page 44)

dye is excited at the appropriate wavelength should be proportional to the quantity of target DNA

present. For each type of fluorescent chemistry, the fluorophore will

emit

a fluorescent light that is

characteristic of the fluorophore used. A range of spectrally distinct fluorophores are commercially

available, introducing the possibility of quantifying multiple targets with different probes in the

same reaction well, otherwise known as

multiplexing

2.3.1 Intercalating dyes

Intercalating dyes bind to the minor groove of double-stranded DNA (dsDNA) producing up to a

thousand-fold increase in fluorescence. As these dyes bind all double-stranded PCR products, this

is a universal chemistry with the major advantage of not requiring the use of a fluorescently

labelled probe. As such, it is a relatively inexpensive approach and one that is ideal as a screening

tool prior to probe manufacture. However, these advantages bring their own disadvantages in that

the ability to bind

all

dsDNA means that both specific and non-specific PCR products, including

primer-dimers, will be reported.

The signal attributable to each PCR product can however be determined by dissociation curve

analysis, a technique that distinguishes PCR products on the basis of melting temperature (Tm).

PCR products of different lengths with different GC contents melt or dissociate at different specific

temperatures. Dissociation curve analysis thus allows the user, via the specific Tm, to establish

that the correct piece of DNA has been amplified. As only a single dye is used to detect all the

products, the chemistry is not open to multiplexing i.e. detection of multiple products.

Mode of action of intercalating dyes:

When the DNA strands are dissociated,

the dye does not bind or fluoresce.

Intercalating dyes bind non-specifically

to dsDNA. When excited, the emitted

fluorescence is relative to the amount of

dsDNA present.

The fluorescence will drop when the

strands start to dissociate. The

temperature at which 50% of the DNA is

single stranded is the Tm. Since the Tm

is influenced by GC content, length and

sequence, it will be indicative of the

identity of a PCR product.

2.3.2 Fluorescent labelled probes

This approach is used when more specific detection of PCR products is required or when there is

more than one target in the reaction tube. Using dye-labelled oligonucleotides that contain a region

complimentary to the target sequence to be amplified, provides advantages in terms of high

specificity and low background. At the same time, the signal is also ensured to be proportional to

the amplified product and not the mass or the length. This chemistry is also multiplex-compatible

such that different ‘coloured’ dyes can be used to report different targets in the same reaction well.

Common examples of fluorescent probe chemistries are detailed below.

2.3.2.1 Hydrolysis probes

This chemistry exploits the 5’ nuclease activity of Taq DNA polymerase to cleave a probe during

PCR. The probes are designed as oligonucleotides that are complementary to a region of the

target located between the upstream and downstream primer binding sites. The probe contains a

fluorophore at the 5’ end and a quencher at the 3’ end; the close proximity of which means that

fluorescence is quenched prior to amplification due to the action of the quencher on the

fluorophore. The quencher absorbs the light emitted by the fluorophore with itself emitting either no

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