The most promising technique found to substitute for Southern blotting was PCR, the basic method invented in 1983 by Kary
Mullis of the Cetus Corporation. PCR is a process where DNA can be artificially multiplied through repeated cycles of duplication
driven by an enzyme called DNA polymerase.
The classical PCR test has advantages of specificity and time over Southern blotting; however, it has limitations. The major
drawback is the lack of accurate quantitative information due to amplification efficiency. In the later cycles of the PCR,
the amplification products are formed in a nonexponential fashion at an unknown reaction rate, and so the link to the initial
quantity in the sample is lost.11
DNA quantification based on conventional PCR relies on endpoint measurements to achieve the maximum sensitivity. At this stage
the reaction has gone beyond the exponential phase, and the resulting correlation between the final product concentration
and the number of initial target molecules is therefore limited.
In 1993 Higuchi et al published an analysis of PCR kinetics; researchers had set up a system to detect PCR products as they
accumulated "real time."12 By 1997 the first high-throughput real-time thermal cycler was on the market; it has revolutionized the detection and quantification
of nucleic acids in many areas of product development.
Table 1. Comparison of Main Validation Criteria Between Southern Hybridization and QPCR
In an evaluation of Southern hybridization and quantitative PCR (QPCR), a comparison of the validation data for each assay
indicated that QPCR is a more robust and accurate method, provided that all controls are in place (Table 1). The following
main advantages of quantitative PCR over Southern blotting outweighed the disadvantages of capital and running costs:
- Assays highly specific for each construct
- Simple 96-well plate format provides high sample throughput
- Rapid assay compared to Southern blotting (one day opposed to one to two weeks)
- Small amounts of DNA are required (<100 ng per assay opposed to 10 _g for the hybridization assay)
- Assay sensitivity is < one copy per cell
- High precision and accuracy
- Safety: no radio-labeling of probes
- No cumbersome manipulation of agarose gels and blots
Assay development time is an important factor in an overall development program. The PCR assay took approximately nine months
and two members of staff to specify and purchase the equipment, and then to develop and validate the test to the ultimate
satisfaction of the authorities. The regulatory bodies then required a large volume of data to compare the two techniques
before they were satisfied that the PCR method could replace the routine Southern blotting method. They also required the
use of both techniques during product release of a number of manufactured batches. As more and more applications now contain
PCR-based measurements, the regulatory authorities are accepting PCR as the de facto standard.
An expample in Which State of The Art Created Confusion
Sometimes the complexity of state-of-the-art methods and their accompanying data lead to inconsistencies rather than increased
accuracy. Unless there are clear benefits from the new tests, regulatory authorities can force a retrenchment to the old tried
and trusted ways. This was the case with a carbohydrate analysis of the same recombinant vaccine development program conducted
in the late 1990s.
The oligosaccharide mapping (N-linked) was performed by fast-atom-bombardment (FAB) mass spectroscopy (MS) and matrix-assisted,
laser-desorption/ionization – time of flight (MALDI-TOF) MS as a characterization test on the active substance. After trypsinization
of the sample, the N-glycans were released by N-deglycosylation with subsequent reverse-phase high-performance liquid chromatography
(rpHPLC) purification. The purified oligosaccharides were permethylated and analyzed by FAB-MS and MALDI-TOF MS, respectively.
Additionally, carbohydrate analysis and total carbohydrate analysis were performed by gas chromatography (GC) MS as a release
test on the drug substance. The glycosidic bonds were cleaved by methanolysis and the released monosaccharides modified to
give volatile trimethylsilyl derivatives, which were subsequently separated by gas chromatography and identified by mass spectrometric
analysis. Identification of individual methylglycosides was based on retention time and mass spectrum by comparison with the
standard mixture. Total carbohydrate was derived by summing the individual monomers.