Trace products, amplification

PCR is a very powerful technique, providing a sizable amount of DNA from a trace of a DNA sample. Hence it would be natural to expect that the trace amount of starting DNA can be quantitated sensitively from the amount of the finally-obtained PCR product. However, this type of quantitation based on the end-point detection is not reliable because of the saturation effects of PCR. This problem has been overcome by real-time PCR, which monitors PCR amplification in real time and enables accurate quantitation from the kinetics of the exponential phase. Real-time PCR thereby provides a highly sensitive and specific quantitation method for nucleic acids. 

The insidious nature of the contamination problem is illustrated by the fact that some contamination fails to be detected by the canonical controls. We (A. Cooper, M. H8ss, and S. PMbo, unpublished observations, 1990) have observed PCR experiments where controls did not show any traces of contamination and the ancient extract gave a clearly visible band. Yet these products proved (on sequencing) to be the result of carryover from a previous amplification. Even multiple controls failed to reveal traces of contamination, and this phenomenon has been observed on several occasions in different laboratories. These observations are consistent with either very low levels of contamination or ancient extracts serving as carriers for low-level contaminants that are not detected in the controls. One control that may be of use is to include an extract of unrelated ancient tissue for... 

Fig. 7.8. Amplification generated by the production of a catalytically active NAD. The main problem with this technique is that even trace amounts of NAD in the enzyme or NADP preparations, will lead to high backgrounds. Alternative amplification methods include enzyme cascades (Blake et al., 1984) and catalyzed reporter deposition (Bobrow et al., 1991).

Whereas detectable PGR amplification of trace amounts of template to provide hundreds of picomoles of modified DNA of the same sequence would have led Perrin et al. to conclude that conditions 2 and 3 for use in selection were satisfied, the inability to produce modified DNA by PGR amplification required additional, albeit indirect, experimental evidence to definitively prove that conditions 2 and 3 could be satisfied. To demonstrate this, Perrin et al. invoked the logical outcome of semiconservative replication over two independent steps (1) the production of modified DNA, which appeared to be sequence specific but could not be proven, and (2) the recopying of modified DNA into unmodified DNA, which if processed correctly would, via a second primer extension reaction, generate a single strand of unmodified DNA with identical sequence to that of the template used in the production of modified DNA. Confirmation of this sequence would, therefore, prove that both conditions were indeed satisfied by... 

Transfer amplification product to a precooled Eppendorf tube, avoiding contamination from the covering mineral oil. (Trace oil can be removed by filtering the sample on an Ultrafree MC Pharmacia filter.)... 

The effect decays with rate l/Tj, so the Ti of the protons in the product must not be very short. Traces of a metal dihydride in equilibrium with H2, even if undetectable by standard NMR, may be seen using PHIP. In a related technique, signal amplification by reversible exchange, SABRE, signal amplifications of 1000-fold are possible. ... 

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