The quantitative analysis of nucleric acid sequences is currently a major issue for basic molecular biology, life sciences industry and many other emerging fields, This type of analytical techniques also represents a wide potential market for specialised products, kits, apparatuses or integrated systems involving both dedicated apparatuses and consumables. Dr Alberto Domingo reports.
There are several methods available for quantitative analysis of nucleic acid sequences, each one having their relative advantages. This type of assay is expected to be specific and sensible: the final quantitative data must reflect the amount of the sequence of interest, without interference from other sequences present in the sample, and also the assay should be able to quantify sequences that are present in very low concentrations.
An extreme accuracy may not be the main issue, since the preprocessing of the real samples usually introduces a considerable error. Conversely, a cost/efficiency evaluation of each method may be quite important, both in terms of consumables, time, and manual requirements per sample, and in terms of investment in equipment and/or reagents for the initial set up. Currently all cost-effective methods are based on amplification reactions, which offer a high sensitivity and reduced manipulations and require a minimal volume of samples. The GeneAmp 5700 (Applied Biosystems) and the LightCycler (Boehringer Mannheim) systems represent two recent instrumental approaches to a PCR-based real time quantitation.
The laboratories with infrequent requirements of such quantitative determinations also need a cost-effective method, in terms of both price and labour, but rarely can take benefit of the investment in an expensive and dedicated apparatus. In this segment of low- to medium-throughput requirements, the clear choice is one of the many variants of competitive amplification methods.
Currently it is probably unnecessary to describe the fundaments of said competitive amplification methods, among which the Competitive PCR is paradigmatic. The key element of such methods is the competitive internal standard. Said standard must be different from the analyte, but both nucleic acid sequences are amplified by the same pair of oligonucleotide primers. Variations in the nature of the standard and in the final step of quantitation originate the diverse flavours of the Competitive PCR assay.
The competitive PCR standard is, by definition, an internal standard of the reaction. In the vast majority of cases, said internal standard is an artificial construct, which is added to the sample, and so it could be defined as exogenous. There are two different groups or categories of these exogenous internal standards.
The homologous competitive standard could be described as a sequence highly related to the analyte, in fact the same sequence with the exception of a short segment which is deleted from, or inserted in, said analyte sequence. These manipulations generate a different but similar sequence, and usually of a different length. The modification is always located outside of the sequences recognised by the primers that drive the amplification.
The heterologous competitive standards, also known as PCR MIMICS, are fully artificial constructs which contain a central heterologous sequence, that is completely unrelated to the analyte, flanked by the same sequences recognised by the PCR primers that amplify the analyte. Again, the standard and analyte sequences are usually of a different length.
The construction of homologous or heterologous competitors is simple, given reasonable experience in molecular biology and laboratory resources. Many research fields that eventually need some quantitative data for nucleic acid sequences not necessarily involve detailed experience in DNA engineering. Constructing homologous competitors may be a hard task in these cases. The construction of heterologous competitors is greatly facilitated by kits available from PanVera, Clontech, and SmartSolutions.
The final differential quantitation of the amplified products from the analyte and the standard sequences may be carried out by different ways. A widely used, reasonably simple and efficient approach is to take advantage of a difference in size by a simple electrophoresis in gel, followed by densitometric analysis of the stained bands. The major disadvantages are the reduced accuracy of the staining and densitometric analysis, and the constant requirement of manual operations.
The Hemicompetitive PCR
The Hemicompetitive PCR represents an original approach, since it focuses the attention on the PCR primers instead of the intervening segments of the analyte and the standard sequences. The Hemicompetitive PCR assay involves an exogenous internal standard sequence plus a set of three oligonucleotide primers. The competition is restricted to one of the three primers, which is common for the analyte and the standard, so it can be referred as the ""common primer"" (oligonucleotide labelled as ac' in Fig. 1). The other two oligonucleotide primers that drive the amplification reaction are different, one being specific for the analyte and the other being specific for the standard, so they can be referred as the aanalyte primer' (aa' in Fig. 1) and the astandard primer' (as' in Fig. 1) respectively.
The analyte and the standard primers are key elements in the Hemicompetitive PCR. It is convenient to carefully design their sequences so that they have a similar theoretical Tm, as well as a clear differential specificity for their respective targets. We find helpful to generate the standard primer as an artificial sequence, by a simple permutation of several positions from the sequence of the analyte primer, so that the base composition is strictly identical in both primers.
A standard sequence for Hemicompetitive PCR, in principle, may be any DNA segment that contains the sequence recognised by the common primer, and lacks the sequence recognised by the analyte primer. The central intervening sequence of the hemicompetitive standard is essentially irrelevant. It can be slightly or completely different to the analyte, or even identical to it, since the differential quantitation step may not depend on this internal sequence at all. Indeed, it is possible to design and set up a minimalist hemicompetitive assay without any central sequence in the analyte or the standard. In this case, the analyte consists in the target sequence of the analyte primer plus the target sequence of the common primer, and the standard segment consists in the target sequences of the standard and common primers.
The Competitive LCR
The Ligase Chain Reaction (LCR) is a powerful sequence-specific amplification method, certainly not as widely known as PCR. In this case the enzymatic reaction is a template-dependent ligation of a pair of oligonucleotides. One strand of the analyte serves as template for a tandem arrangement of two oligonucleotides, which are then ligated by a thermostable DNA ligase. The separation of the template strand and the newly formed ligation product now produces two sequences that behave as templates for two new ligation events, which in turn generate four templates for four other ligations, and so on. The LCR involves a set of four oligonucleotides, each two of them complementary to one strand of the analyte. The core idea of the Hemicompetitive PCR can be directly applied to LCR to generate a new quantitative assay, the Competitive LCR, which involves an exogenous internal standard plus a set of six oligonucleotides.
The competition is restricted to two of the oligonucleotides, which are common for the analyte and the standard, so they can be referred together as the acommon pair' (ac1' and ac2' in Fig. 2). The other four oligonucleotides comprise the aanalyte pair' (aa1' and aa2' in Fig. 2) and the astandard pair' (as1' and as2' in Fig. 2) which are specific for the analyte and the standard respectively.
The design of a Competitive LCR assay is greatly facilitated by using the same approach of generating the standard pair as artificial sequences by a simple permutation of several positions from the sequences of the analyte pair, so that the base composition is strictly identical in both pairs.
In a Competitive LCR assay the standard consists in the target sequence of the standard pair plus the target sequence of the common pair, in the same way as the analyte is defined by and consists in the target sequences of the analyte and common pairs. As a mater fact the whole system is completely equivalent to the above described as the minimalist Hemicompetitive PCR setup. In both cases the standard sequences are short enough to be directly generated by chemical synthesis.
A common advantage of both methods is that any label introduced in the oligonucleotides becomes incorporated in the amplified products of the analyte and the standard. A label in the common primer or in one or both oligonucleotides of the common pair, for example with biotin or other capture moiety (depicted as an anchor in the figures), will be present in both types of amplified products.
Two different labels in the analyte and standard primers or pairs, for example two detection moieties like fluorescent groups with different spectral characteristics (suggested by the flags in the figures), will be incorporated differentially in the two types of amplified products.
The combination of a common capture labelling plus a differential detection (and quantitation) labelling, together as a direct result of the amplification reaction, makes possible or simplify the quantitation step.
Currently there are many different methodological as well as instrumental solutions for the sequence specific quantitation of nucleic acids. Each one has its own advantages and is better adapted to a specific segment of users. The Hemicompetitive PCR and the Competitive LCR assays are two of our contributions to this field.
Dr. Alberto Domingo is associate professor of the Department of Biochemistry and Molecular Biology, University of Alcalá, Alcalá de Henares, Madrid. Fax: +34 91885 4585. Rocío Vega is his personal research technical assistant. Gerónimo Fernández, Miguel Moreno, Esther Rincón, Sonia Jimenénez and Sergio Ruiz are members of his research group.
