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

Clinical Laboratory Science, Fall 2005 by Zhao, Richard

There are two types of probes used in real-time PCR: hydrolysis probes for real-time quantification and hybridization probes for single nucleotide polymorphisms (SNPs) and mutation detections. Examples of hydrolysis probes include TaqMan probes, molecular beacon probes, and scorpion primer-probes.7,8 The TaqMan probes present as linear oligonucleotides; oligonucleotides of molecular beacon probes are typically in a hairpin format labeled at one end with a quencher and at the other end with a fluorescent reporter. In the absence of gene targets, the fluorescence is quenched. However, when molecular beacons hybridize to their gene targets, the hairpin structures open and they emit intense fluorescent signals for real-time detections. Scorpion primer-probes incorporate both the primer and probe into oligonucleotides that exist in a hairpin loop structure. Similar to molecular beacons, the fluorescence of scorpion primer-probes is normally quenched. Upon primer-mediated DNA synthesis of the gene targets, the scorpion probes hybridize to the newly formed complementary sequences, separating the fluorescent reporters from the quenchers thus restoring the fluorescence. In principle, molecular beacons and scorpion probes provide better probetarget binding specificity than linear probes.

Hybridization probes have been used for detections of mutations and SNPs by combined use of melting curve analysis and sequence-specific fluorescence resonance energy transfer (FRET). In a FRET melting curve analysis, two hybridization probes that are complementary to a continuous region of the gene target are labeled with fluorescein or LC Red 640, respectively. When the two fluorescent labeled probes come together upon gene-specific hybridization, excitation of fluorescein causes an energy transfer, which subsequently results in excitation of the LC Red 640 molecule. Such a FRET allows detection of specific probe-gene hybridization. Melting curve analysis is based on the fact that each double strand DNA has its own unique melting temperature (Tm). A single change in nucleotide will result in change of Tm. Therefore, combined use of FRET for gene-specific hybridization with mutation-specific changes of Tm enables detection of specific gene mutations with high accuracy.

Nucleic acid sequence based amplification (NASBA)

NASBA is another target-based amplification method that is formerly known as 3SR (self-sustaining sequence replication). In contrast to PCR, however, this method is essentially an in vitro version of the natural replication of retroviral RNA. Therefore, this assay is useful only for detecting RNA targets such as HIV or HCV. A standard NASBA reaction contains T7 RNA polymerase, RNase H, avian myeloblastosis virus (AMV) reverse transcriptase, nucleoside triphosphates, two specific primers, and appropriate buffer contents. For quantification of HIV viral load, for example, primer 1 is about 45 bases in length with an average of 20 bases at the 3' end that are complementary to the 3' side of the target sequence. The 5' end of this primer contains a promoter sequence that is recognized by T7 RNA polymerase. Primer 2 is about 20 bases in length and is derived from the opposite (5' direction) side of the target sequence. The NASBA test involves repetitive reverse transcription from an RNA template by AMV reverse transcriptase and RNA amplification from cDNA by gene transcription viaT7 promoter. Approximately 40 copies of RNA can be made (versus two copies/cycle in PCR) in each cycle for each copy of the RNA target. Within a 90 minute reaction, approximately 1012 RNA molecules can be made starting with 10 copies of purified RNA molecules.