Probe amplification methods

Genomic quantification is necessary to identify large deletions or duplications that occasionally cause single gene disorders. A novel molecular method (multiplex ligation-dependent probe amplification, MLPA) has been recently developed for this purpose and is presented in this chapter. 

Nucleic acids are also valuable as diagnostic reagents. In combination with the recently developed amplification method, polymerase chain reaction (PCR), nucleic acid probes allow the detection of trace amounts of a specified nucleic acid sequence. Thus, the presence of the DNA of pathogens can be ascertained with great sensitivity and rapidity. Further, these tools permit the analysis and the detection of gene defects in individual patients, and aid in the tailoring of therapeutic strategies. These applications are discussed in Chapter 8. 

One advantage that PCR has over many other DNA probe diagnostic methods is that small, degraded, damaged, and unpurified DNA can still serve as a template for the first cycle of amplification. Because subsequent cycles mainly use the newly synthesized product of previous cycles as template, poor-quality targets are irrelevant once amplification is underway. 

The first detection methods used with PCR were radioactively labeled probes to identify specific amplified sequences (M8, SI). With improvements in amplification specificity it became possible to visualize amplified DNA of the predicted size directly by its fluorescence on an agarose or polyacrylamide gel (M9) following staining with ethidium bromide. Probe-based methods remain a key feature of current detection systems primarily because of the additional information and sequence specificity they provide. Probes have been converted to nonisotopic colorimetric systems (B6) by labeling them with an enzyme such as... 

Figure 8.13 Schematic description of a triple amplification method for microRNA sensing based on (i) DNA-microRNA recognitions, (ii) boronic acid attachment of gold nanoparticles with biotin, (iii) binding streptavi-din with phosphatase, and finally (iv) release of the redox active probe. (Reproduced from ref. 95 with the permission of Elsevier.)...

A diagnostic method using fluorescence labeled DNA probes to detect and quantify the number complementary chromosomal sequences on a cellular resolution. A related technique that also allows assessment of gene amplifications, but without precise quantification of copy numbers is the chromogenic in situ hybridization (CISH). Here, instead of a fluorescent dye an enzyme that can generate a colored precipitate in the tissue samples is coupled to the DNA probe. 

---