Mutant and chimeric DNA sequences are often useful for analyses of the structure and function of genes and proteins. Mutations can be introduced by using PCR primers that incorporate one or more point mutations, deletions, or insertions. Two or more distinct DNA sequences can be joined using splice overlap extension PCR.5 This method, which does not require the presence of restriction sites of other specific sequences, greatly facilitates the production of cDNAs encoding chimeric and fusion proteins.

QUANTIFICATION OF mRNA USING PCR

PCR has been widely used to measure the level of expression of mRNAs. The major advantage of PCR based methods of mRNA quantification is high sensitivity. PCR based methods may be the only option if the amount of starting material is limited (as often occurs when analysing mRNA expression in primary cells) or if the target sequence is expressed at very low copy number. However, when RT-PCR is carried out in standard fashion (reverse transcription of RNA followed by a fixed number of cycles of cDNA amplification), the amount of product DNA does not relate in a consistent or predictable manner to the amount of input RNA. There are several reasons for this. For example, there may be contaminants in the RNA preparation that interfere with reverse transcription or amplification, and oligonucleotides will differ greatly in their ability to specifically prime product synthesis. Another major problem is that the efficiency of amplification varies from cycle to cycle. In theory, the amount of PCR product should double each cycle, but in practice this is not the case and the efficiency of amplification drops considerably in later cycles. Two approaches have been widely used to make RT-PCR a quantitative technique: competitor PCR and real time quantitative PCR.

In competitor PCR a competitor DNA construct is added to each PCR reaction (fig 1). The competitor construct is identical to the authentic sequence except for the addition (or deletion) of a portion of the target sequence located somewhere between the two amplification primers. Amplification results in two products—an authentic cDNA product and a competitor product that is somewhat larger (or smaller) than the authentic product. Since the efficiency of amplification of both products should be similar in each cycle, the ratio of authentic product to competitor provides a good quantitative (or at least semiquantitative) estimate of the amount of input cDNA. Expression of specific mRNAs can be normalised by comparison with one or more “housekeeping” gene. Competitors are available for an assortment of genes and can be readily produced by various methods.6 7The method is well suited for analysis of the expression of a small to moderate number of mRNAs. For example, competitor PCR was used to analyse the expression of cytokine mRNAs in a murine model of asthma (fig 2).8 Careful titration of input cDNAs and competitors may be required, especially to produce more rigorous quantification, and the method may be quite labour intensive, especially when large numbers of samples are to be analysed.

A second approach to quantitating mRNAs is real time quantitative PCR. This method involves the use of a specialised thermal cycler capable of measuring fluorescence in each reaction during each cycle. A fluorescent signal is produced either by using a dye (SYBR Green) that fluoresces after binding to double stranded DNA, or by using a special oligonucleotide with a fluorescent reporter dye that is released by the DNA polymerase during each extension cycle. The amount of input cDNA is quantified by identifying the “threshold cycle”, the first cycle in which product can be unambiguously detected. Lower threshold cycle numbers are associated with higher amounts of input cDNA. Since PCR products are detected early, problems attributable to non-exponential amplification are minimised. This method can be made highly reproducible and is especially well suited for the measurement of one or a few mRNAs from a large number of sources, as may be required for screening the effects of libraries of compounds. It does, however, require a relatively expensive specialised cycler and careful development and testing of appropriate oligonucleotide primers for each mRNA.

Difference analysis

Finding genes/proteins that are differentially expressed in diseased tissues or altered cells is a key to understanding the pathogenesis, aetiology, and/or response of diseases to treatment. Difference analysis is more readily achieved by examining mRNA differences than protein differences, largely for technical reasons—it is substantially easier to identify differentialgene expression thanprotein expression in disease tissues. The early approaches to analysis of differential mRNA expression relied upon subtractive hybridisation, an approach that has detected the expression of a number of important genes despite the time consuming and labourious nature of this approach. It does not detect mRNAs which are expressed at very low levels. As there are up to 100 000 human genes and a single cell expresses perhaps 25 000 of these as distinct mRNA transcripts, with 98–99% being rare, the ability to detect differences in expression of mRNA at low levels is crucial.

DIFFERENTIAL DISPLAY

Differential display PCR and its related technologies involve the RT-PCR amplification of two different mRNA populations using defined oligomers followed by separation of the resulting fragments side by side on a denaturing polyacrylamide gel.9 10Differentially expressed bands at different points on the gel are removed and re-expanded by PCR and cloned. A number of variations of this technology have been developed to try to overcome the main problems—namely, the labourious nature of the approach, the generation of false positive bands, and the limited ability to quantify levels of mRNA expression. It is available in kit form.

REPRESENTATIONAL DIFFERENCE ANALYSIS (RDA)

RDA is an alternative method which is aimed at reducing the number of candidates genes. It combines a subtractive hybridisation step with amplification using PCR, thus optimising for differences.11-13 Again, a number of variations have been developed.

SERIAL ANALYSIS OF GENE EXPRESSION (SAGE)

SAGE was developed by Kinzler, Vogelstein and Velculescu and involves the isolation of unique 3′ sequence tags which are then concatenated and sequenced.14 15 Thus, each individual clone that is sequenced contains many partial length tags (about 10–50). Importantly, the frequency of many transcripts and the starting mRNA pool can be determined by the frequency with which a specific sequence tag is found in the sequenced population. SAGE has advantages in terms of efficiency, quantification, the ability to detect novel genes and, importantly, the ability to detect low abundance transcripts (table 1).

Advantages and limitations of SAGE and DNA chip technology for analysis of difference in mRNA expression in different tissues

The success of SAGE largely relies on the availability of sequences representative of genes in the particular target species. Such genes are largely in the form of expressed sequence tags (ESTs). There are now a large number of ESTs in public and private databases. Approximately 60% of human genes are now currently available while a lesser proportion of murine genes are available on database. There are also some large public and private “warehouses” containing large scale genetics databases—for example, Incyte Genetics.

The application of this technology to two different pulmonary tissues/samples/conditions may run the risk of producing large numbers of sequence differences without there being a readily available database to analyse the significance of these differences. Thus, it is possible to generate a large amount of information that is, at this stage, difficult to interpret.

DNA chip arrays in that novel genes can be discovered and more sequences can be analysed. SAGE can be used in multiple experiments. Genzyme has acquired the commercial rights to SAGE technology. SAGE analysis of lung cancer has recently been published.

Editorial Team
Journal of Molecular Biology and Biotechnology
London, United Kingdom
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