Enhanced Diagnostic Tools
Initially, intercalator dyes were used to measure real-time PCR products. The primary disadvantage to these dyes is that they detect accumulation of both specific and nonspecific PCR products.
Development of TaqMan Chemistry
Real-time systems for PCR were improved by the introduction of fluorogenic-labeled probes that use the 5´ nuclease activity of Taq DNA polymerase. The availability of these fluorogenic probes enabled the development of a real-time method for detecting only specific amplification products. The development of fluorogenic labeled probes also made it possible to eliminate post-PCR processing for the analysis of probe degradation.
How TaqMan Sequence Detection Chemistry Works
The TaqMan chemistry uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR.
Two Types of TaqMan® Probes
Applied Biosystems offers two types of TaqMan probes:
TaqMan® MGB Probes Recommended for Allelic Discrimination Assays
Applied Biosystems recommends the general use of TaqMan MGB probes for allelic discrimination assays, especially when conventional TaqMan probes exceed 30 nucleotides. The TaqMan MGB probes contain:
Consequently, the TaqMan MGB probes exhibit greater differences in Tm values between matched and mismatched probes, which provide more accurate allelic discrimination.
Advantages of TaqMan® Chemistry
The advantages of the TaqMan chemistry are as follows:
Disadvantages of TaqMan® Chemistry
The primary disadvantage of the TaqMan chemistry is that the synthesis of different probes is required for different sequences
SYBR Green I Dye Chemistry
Small molecules that bind to double-stranded DNA can be divided into two classes:
Regardless of the binding method, there are two requirements for a DNA binding dye for real-time detection of PCR:
Applied Biosystems has developed conditions that permit the use of the SYBR Green I dye in PCR without PCR inhibition and increased sensitivity of detection compared to ethidium bromide.
How the SYBR Green I Dye Chemistry Works
The SYBR Green I dye chemistry uses the SYBR Green I dye to detect polymerase chain reaction (PCR) products by binding to double-stranded DNA formed during PCR. Here’s how it works:
Advantages of SYBR Green I Dye
Disadvantage of SYBR Green I Dye
The primary disadvantage of the SYBR Green I dye chemistry is that it may generate false positive signals; i.e., because the SYBR Green I dye binds to any double-stranded DNA, it can also bind to nonspecific double-stranded DNA sequences.
Another aspect of using DNA binding dyes is that multiple dyes bind to a single amplified molecule. This increases the sensitivity for detecting amplification products. A consequence of multiple dye binding is that the amount of signal is dependent on the mass of double-stranded DNA produced in the reaction. Thus, if the amplification efficiencies are the same, amplification of a longer product will generate more signal than a shorter one. This is in contrast to the use of a fluorogenic probe, in which a single fluorophore is released from quenching for each amplified molecule synthesized, regardless of its length.
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