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January 01, 2011 (Vol. 31, No. 1)

Improving Diagnostics-Related Informatics

Completion of the Human Genome Project Has Upped the Ante

  • Innovative Analytical Tools

    Over the last four decades, a number of revolutionary analytical tools have been developed, including restriction enzyme digestion, hybridization, and PCR. Although these tools were used in the HGP, the brunt of the work was handled by Sanger sequencing. Sanger sequencing is not a panacea though; its high cost as well as the need to sequence more than one target severely limits its use in the clinical testing market.

    PCR was developed primarily to produce short segments of double-stranded DNA, mainly for cloning purposes. It is able to generate millions of copies of short genomic segments, which has led to it largely replacing the traditional culture method.

    Restriction fragment length polymorphisms (RFLP) rely on gel-electrophoresis analysis. Even though it is sensitive, its use of radio-isotopes has limited its potential for extensive use in clinical testing. Nucleic acid amplification techniques have been shown to have nonspecific amplification and potential inhibition by materials in clinical samples.

    In order to adapt PCR to clinical settings, a number of factors needed to be resolved. When PCR is performed in a single tube, the optimum reaction condition is compromised. Further, the key enzyme, Taq polymerase, needs only two pairs of nucleotides to anneal for its 3'–5' polymerization. This nonspecific polymerization could occur during ramping down from the denaturing step, which usually results in nonspecific amplification.

    Amplification is a key part of the process as it is important to identify what is amplified. The identification step can be strengthened through the use of two labeled-probe systems. Clinical samples may, however, have more than two targets. The inability of the traditional PCR/labeled combination to fulfill this clinical need gave rise to chip technology, which is an extension of the Southern blot. Another method is focused on detection in narrow wavelengths; an example is the Luminex platform.

    Real-time PCR sprung out of the “hot-air” thermocycling principle in which the dynamics of amplification are monitored graphically. The basic principle of the real-time PCR system is its hybridization of labeled probe to the target.

    Platform technologies that are presently used in routine diagnostics are mainly for detection, indicating either the presence or absence of the intended target. These technologies have been validated by correlation to clinical symptoms and/or prior accepted assays.

    There are, currently, only a few FDA-approved tests, and laboratory-developed tests (LDT) are mostly used for routine clinical testing. Almost all LDTs use surrogate technologies such as real-time PCR, FISH, and mass spectrometry where the results are generated in a format that cannot be verified. Hence, the culture/ELISA combination method still continues to be in demand in the clinical market.

  • Striving for Clinical Use of Sequencing

    Because of the limitations of existing technologies, it makes sense that firms are attempting to bring sequencing platforms into routine clinical use. NGS can generate short sequences through random priming/termination. This fact makes whole-genome sequencing possible.

    Next-generation sequencers are mainly used in research settings, primarily for the discovery of SNPs and their potential correlation to diseases. Capillary electrophoresis based sequencing is also being retooled with multiple amplicon sequencing by MultiGen in an attempt to make it suitable for routine testing. Generation of dia-informatics for routine testing will enable better patient management and drug development. Success of these approaches will depend upon the cost and comprehensive clinical utility.

Posted 1/3/2011 by Wouter

"The late Fred Sanger"? That may be a little premature.


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