Primer based assays

The three main categories of hybridization probes for real-time PCR are (1) cleavage based assays such as TaqMan, (2) displaceable probe assays such as Molecular Beacons and (3) probes which are incorporated directly into primers such as Scorpions. 

Quantitative PCR has been widely used to determine the amount (number of molecules) of DNA molecules in a test sample. The best quantitative PCR method involves the addition of known amounts of a similar DNA or RNA fragment, such as one containing a short deletion or specific mutation, to the test sample before amplification. Such internal standards must be precisely calibrated to ensure that they are amplified and detected in a form and manner that are similar to the test sample. The ratio of the internal standard and the targeted template will depend on the amount of internal standard added and allows for the determination of the amount of the targeted molecule in the test sample. Therefore, the ideal standard for quantitative amplification based assays should have a structure that is comparable to the template of interest and which allows for the simultaneous amplification of both template and standard using a single primer pair. 

It is apparent that signal amplification provides increased sensitivity over direct labeling. This is especially true for fluorescent-based assays. One of the most sensitive signal detection technologies is the immunoRCA (Schweitzer et al., 2000). Rolling circle amplificahon (RCA) is combined with antibody detection. RCA involves the amplification of circularized oligonuceotide probes under isothermal conditions by DNA polymerase (Lizardi et al., 1998). With immunoRCA, the 5 primer is attached to the reporter antibody. Initiation of the amplification starts when circular DNA template binds to the attached primer. 

The first PCR-based assay for detection of A. niger used sequences from the 18S rRNA gene as the target for primer binding (J imenez etal.,1999). The authors used primers originally published by Melchers et al. (1994),... 

The primer extension assay is similar to the dideoxy method commonly used in sequence analysis. In the primer extension assay, a region containing the mutation to be assayed is amplified in a first PCR reaction. A specific primer, which is designed to anneal directly upstream of the base which is the site of a known mutation, is then used in a second reaction. Radioac-tively labeled dideoxy nucleotides corresponding to either the normal or the mutant base at the potential mutation site are added in separate tubes. During the reaction, the labeled nucleotide added to the 3 end of the primer will depend on the sequence at the potential mutation site. If the normal sequence is present at the potential mutation site, only the reaction containing the normal labeled dideoxy nucleotide will produce a labeled primer. Conversely, if the mutant sequence is present at the mutation site, only... 

Another report reveals, however, that in vitro DNA-binding assays are insufficient to predict platinum antitumor activity [15]. Primer extension (Fig. 1) was used to identify specific adducts formed by platinum complexes on DNA in HeLa cells. The DNA adduct profile correlated well with in vivo antitumor activity for cis- and trans-DDP, Pt(en)Cl2, and two acridine-tethered platinum complexes. When the complexes were allowed to react with purified DNA in solution, there were no substantial differences in adduct profiles between active and inactive compounds. This result demonstrates that cell-based assays can be better predictors of in vivo activity than in vitro assays, particularly when the in vitro screen does not require aunique, mechanism-based molecular interaction. 

Fig. 2. Primer extension assay to detect DMS-modified nucleotides in single-stranded RNA. RNAs modified with DMS are examined by primer extension. Bases at which modifications occur are unable to form complementary hydrogen bonds with the cognate nucleotide. Thus, chain termination results. Primer extension products (PE) terminate one nucleotide before the modified residue compared to a sequencing ladder.

Genotype is scored for each sample based on which of the allele specific amplifications were positive. Specificity is determined by the amplification kinetics of each assay and the differential efficiency of the polymerase in extending 3 matched and 3 mismatched primers. This assay format fails to multiplex and requires significant optimization of each assay, but uses cheap unmodified oligos and relatively cheap instrumentation. 

Although a number of quantitative assays are available for allele frequency estimation, most are difficult and expensive to develop or implement. In this chapter, we describe the most versatile and least expensive assays for allele frequency estimation, namely, the template-directed dye-terminator incorporation assay with fluorescence quenching detection (the FQ-TDI assay [3]). The FQ-TDI assay is a real-time homogeneous primer extension assay based on two principles, namely, that deoxyribonucleic acid (DNA) polymerase catalyzes the allele-specific incorporation of a dye-terminator at the polymorphic site and that the fluorescence intensities of a fluorescent dye decreases significantly when it is incorporated into primers. 

In general terms, DNA-based assays are easier to develop than immunoassays since the only requirements are the availability of the gene or DNA sequence to be targeted. These sequences are needed to design primers (PCR) and probes (RT-PCR) as well as for determining the G/C ratio to optimize assay conditions. This information can be obtained from databases, and software is available to facilitate the design of primers and probes. These probes have also become less expensive to produce. 

As an alternative to this approach, one can directly measure dNTP analog incorporation and extension using a gel-based assay and a DNA template consisting ofaS - P -labeled primer, annealed to a single-stranded template (as an example, see ref. 44). Primer extension in the presence of three dNTPs produces a block that can be overcome with the addition of incorporable analogs with extensible 3 -OH termini. 

Primers The primers are short (15-30) oligonucleotide sequences designed to base pair or anneal to complementary sequences that flank the DNA target sequence to be amplified. The primers are added at 0.1-1 qM in the assay. 

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