Quantitative Real-Time PCR

Q-PCR is a PCR based technique suitable for quantitative analysis of target organims in enrichments or other samples. With sampling in time the Q-PCR technique can provide very interesting data of the development of a microbial community in time. With specific primers targeting ANME1, ANME2c, DSRB, Eubacteria and Archaea a quantitative analysis can be performed. The outcome can be presented as absolute cell numbers or relative cell numbers.

Different aspects of working with Q-PCR analysis on environmental samples will be discussed. In the Forum a separate topic about Q-PCR will be availible for more detailed questions.

1. Melt curve analysis (SYBR GREEN)

When you make an exponential number of copies through 30-40 cycles of PCR, you end up with a lot of copies of the same amplicon. Let's use a 500 bp amplicon as an example. If your original template contains the DNA you are looking for, from one copy you will make 2 copies, then 4, then 8, 16, 32, 64, so on and so forth. In traditional PCR you take this collection of amplicon and run it out on a gel. If multiple copies of this 500 bp amplicon have been generated, it will be visualized as a band at 500 bp.

SYBR Green Q-RT PCR works in a somewhat similar way. When you make an exponential number of the same amplicon, you can increase the temperature slowly and wait for fluorescence to be lost.

The temperature at which DNA melts depends on several specific characteristics, making DNA melting temperature (Tm) curve analysis a useful tool in distinguishing closely related DNA fragments. Melting of double-stranded DNA (dsDNA) occurs when the two complimentary strands separate during denaturation. Separation of strains results from broken hydrogen bonds. Because complimentary bases adenine (A) and thymine (T) form two hydrogen bonds while complimentary bases guanine (G) and cytosine (C) form three hydrogen bonds, the nucleotide content can significantly affect the Tm. In addition, nucleotide sequences with higher ratios of guanine and cytosine (GC content) have a higher Tm due to stacking interactions between neighboring base pairs. DNA fragment length also contributes to thermal stability. Longer DNA fragments (>1000bp) may have internal melting domains that result in complex Tm curves. Generally, a single transition temperature peak occurs within a Tm curve of shorter fragments, although complex Tm curves are possible. The Poland algorithm has been employed to determine the thermal stability of a piece of dsDNA using base stacking and denaturation behavior of dsDNA fragment. This results in a predicted transition curve for that piece of dsDNA interpreted as a Tm curve.

Tm curve analysis using SYBRŽ Green technology provides qualitative measurements capable of distinguishing between DNA fragments. SYBRŽ Green is a standard fluorescent dye that binds to the minor groove of the DNA double helix. In an unbound state, when little or no dsDNA is present, the level of SYBRŽ Green fluorescence is relatively low. However, upon binding to dsDNA the level of fluorescence is greatly increased, up to 1000-fold. Therefore, in a PCR reaction, as more cycles are completed and copies of the DNA template are exponentially produced, the amount of fluorescence detected also increases exponentially. This is the operating principle behind QRT PCR, a measurable fluorescent signal corresponds to the amount of amplification product present.

A subsequent melt curve analysis following the PCR reaction also takes advantage of the DNA binding properties of SYBRŽ Green. Similar to how the SYBRŽ Green fluorescence increases with higher levels of amplification product, SYBRŽ Green fluorescence decreases during the breakdown of this dsDNA product. As the temperature is increased through the dissociation temperature, the dsDNA product separates into complimentary strands. As a result, SYBRŽ Green molecules are released, drastically decreasing fluorescence. The Tm is calculated to be the point at which maximum fluorescence is lost. Divergent DNA fragments with as little as a single bp change could theoretically be identified by examining these Tm profiles. Unfortunately the Q-PCR technique is still poorly understood in this specific field and data interpretation is still subject to personal view of the researchers.