From Single Cell Gene-based Diagnostics to Diagnostic Genomics: Current Applications and Future Perspectives

Clinical Laboratory Science, Fall 2005 by Zhao, Richard

Molecular diagnostics is a branch of clinical diagnostics that uses primarily DNA or RNA as a biomarker for clinical testing. It combines various gene-based amplification technologies with highly sophisticated detection methods for the clinical diagnosis of a vast variety of diseases including infectious diseases, cancer, and inherited diseases. The principal application of gene-based amplification technology is to identify pathogen or gene-specific nucleic acid sequences that are used as surrogate markers for the identification of either infectious pathogens or alteration of disease-related genes. There are generally three classes of gene-based amplification technologies: target-based, e.g., PCR; probe-based, e.g., LCR; and signal-based, e.g., bDNA. Real-time detection of PCR allows us to quantify amplified amplicons with a broad dynamic range and it offers a unique way to detect genetic mutations. Other technologies such as immuno-PCR and bio-barcode assay (BCA) combine different amplification tactics offering extreme detection sensitivity ranging from femtogram (10^sup -15^) to zeptogram (10^sup -21^). Even though quantum dots technology is in its infant stage, its potential to further increase diagnostic sensitivity and specificity is likely beyond our current imagination. Future diagnostic technologies include the use of genomic and proteomic approaches especially in pure cell types or even in the single-cell level, which open up endless new possibilities for gene-based diagnostics at entirely different levels. In this article, principles of various current gene-based amplification and detection technologies along with their clinical applications are discussed. New technologies that could potentially be used in future gene-based diagnosis are introduced.

ABBREVIATIONS: BCA = bio-barcode amplification; bDNA = branched DNA; FRET = fluorescence resonance energy transfer; IPCR = immuno-PCR; LCM = laser capture microdissection; LCR = ligase chain reaction; NASBA = nucleic acid sequence based amplification; PCR = polymerase chain reaction; qdots = quantum dots; SDA = strand displacement array; SNP = single nucleotide polymorphism; T^sup M^ = melting temperature.

INDEX TERMS: gene-based amplification; molecular diagnostics.

Clin Lab Sci 2005;18(4):254

LEARNING OBJECTIVES

1. Describe the classification of gene-based amplification methods.

2. State the principles of gene-based amplification methods.

3. Discuss the advantages and disadvantages of gene-based diagnostics.

4. Define future prospectives of gene-based diagnostics.

Twenty years has passed since the first description of the polymerase chain reaction (PCR) technique and its application to amplification of the β-globin gene sequence and to restriction fragment length polymorphism analyses for the diagnosis of sickle-cell anemia.1 During this period of time, there has been a nearly explosive growth in the number and variety of new gene-based methodologies in the field of genebased diagnostics, also called molecular diagnostics.

Molecular diagnostics, based on nucleic acid amplification and detection technologies, is a branch of clinical diagnostics that uses primarily DNA or RNA originated either from patients or pathogens as the biomarker for the clinical testing. These genebased diagnostic approaches span from detection of infectious diseases, cancer detection, and genetic diseases to forensic identity testing. One of the most desirable features of gene-based technologies in clinical diagnostics is their rapid, specific, and direct detection of genes of interest with very high sensitivity. Many of them allow additional and precise quantification of genes of interest with broad dynamic range. Importantly, they can be performed directly from clinical specimens without the need for laboratory cultivation, which is often time-consuming.

A unique feature of gene-based diagnosis of infectious diseases is that it can detect pathogens that cannot be detected by conventional diagnostic tests. For example, we can now readily detect many of the viral infections that are either difficult or impossible to culture in the laboratory. Another example of using gene-based assays is that we can now detect HIV infection in newborns on the day of birth. Previously, when antibody-based tests were used, a positive diagnosis was not possible until three to seven months of age. In another article in this Focus section, Drs Niel Constantine and Richard Zhao from University of Maryland School of Medicine describe in detail the molecularbased diagnosis of HIV-1 infections.

Molecular testing can also be used to predict potential risk of an individual for onset of a specific cancer such as breast cancer with predispositions oibrcal/2 gene mutations.2·3 Dr W Craig Hooper and his colleague Stacy C League from the Centers for Disease Control and Prevention provide a detailed description of this type of testing in congenital thrombosis in this issue. If clinical symptoms or other diagnostic tests implicate a specific cancer such as T-cell leukemia, gene-based testing could also be used to confirm diagnosis by detecting possible known T-cell gene arrangements. Furthermore, these same tests could be used to monitor success of anti-cancer therapy. For example, disappearance of the same T-cell gene re-arrangement would indicate cancer remission in the patient.