Scibiochem: PCR sizing and Triplet-primed PCR (TP-PCR)

PCR sizing and TP-PCR are both PCR-based techniques which can be used in the diagnosis of single gene disorders.

The main difference between the techniques is that PCR sizing is size-limited in what it can detect, whereas TP-PCR gets round this with the addition of an extra primer.

PCR sizing

PCR sizing can be used to detect a small deletion or insertion mutation.

An example is the detection of the 3-base-pair deletion that results in loss of a phenylalanine in the CFTR protein (ΔF508) which is the most common mutation causing cystic fibrosis (CF) in the Caucasian population.

PCR sizing is also used in testing for the triplet-repeat expansion diseases, eg Fragile X, Huntington’s disease, myotonic dystrophy etc.

The use and limitations of PCR sizing is well exemplified by Fragile X testing (for more information about this condition see the section on Southern blotting).

PCR sizing is able to detect normal and premutation FMR1 alleles, but the expansions seen in affected individuals may be too big to detect and further testing using other methods is required.

Figure 1 shows the results of Fragile X PCR sizing:

Diagram showing the results of Fragile X PCR sizing

Figure 1: Results of a Fragile X PCR sizing
© St George’s, University of London

In the top trace, there is a peak at around 31 repeats, representing a normal allele.

If you look closely however you will also see a further series of small peaks around 88 repeats, shown at greater magnification in the trace below. This series of peaks represents a premutation allele.

The instability of this allele can be seen by the range of peaks, with a median size of 88 repeats.

This result, therefore, shows a female fragile X premutation carrier, with one normal allele and one unstable premutation allele.

Whilst in some scenarios, PCR sizing alone can provide a result, full expansions are too big to detect with PCR sizing and require Southern blotting or triplet-primed PCR.

This is also the case for testing of females with a family history suggestive of fragile X, or for prenatal testing, as there could be mosaicism, which PCR sizing alone would not detect.

Triplet-primed PCR (TP-PCR)

Triplet-primed PCR (TP-PCR) is a technique which is replacing Southern blotting for FMR1 analysis in the majority of laboratories. It is quicker and less labour-intensive, taking 4 hours rather than 4 days, which is obviously highly desirable in the prenatal setting.

Unlike Southern blotting, it requires very little DNA. It is however very expensive since the company producing the kit currently has a monopoly making its widespread introduction into clinical laboratories challenging.

The main limitation of standard PCR sizing is that it may fail to detect full-size expansions. Triplet-primed PCR overcomes this by the addition of an additional reverse primer (P3, shown in Figure 2), which is complementary to the (CGG)n repeat. The P1 and P2 primers flank the CGG repeat region, whereas the P3 primer binds all along the CGG repeat.

Image showing the Triplet-primed PCR process and how it overcomes the main limitation of standard PCR sizing

Figure 3 shows a TP-PCR result for a full mutation. In this case the P1 and P2 primers have successfully amplified the full-length >200 CGG repeat region (shown by the peak to the right of the figure). In some cases however the full-length product may be too large to amplify and could therefore be missed. However with TP-PCR, its presence would be indicated by the typical profile of products from the P1 and P3 primers (the series of CGG primed amplicons, up to around 200 CGG, to the left of the figure).

Graph depicting a Triplet-primed PCR result for a full mutation

A further advantage of TP-PCR is that it allows mapping of possible AGG elements (known as AGG ‘interruptions’) within the CGG repeat region.

The number and spacing of AGG elements is increasingly being recognised as important as they may affect the stability of the repeat, and hence the risk profile in fragile X carriers.

The table below summarises the main differences between Southern blotting and TP-PCR, and the relative pros and cons of each method for Fragile X testing: