Question

DNA detection methods

Answer 1

Methods for DNA sequence identification in real time and with high sensitivity are of great scientific and economic interest (1–3). Their applications include medical diagnostics, identification of genetic mutations, gene delivery monitoring, and specific genomic techniques (4). Cationic organic dyes, such as ethidium bromide and thiazole orange, emit when intercalated into the grooves of double-stranded DNA and serve as direct DNA hybridization probes, but lack sequence specificity (5, 6). Energy/electron transfer chromophore pairs for strand-specific assays exist, but require chemical labeling of two nucleic acids or dual modification of the same altered strand (i.e., molecular beacons) (7, 8). Difficulties in labeling two DNA sites result in low yields, high costs, and singly labeled impurities, which lower detection sensitivity (9). Much of the motivation behind improving DNA sensing is to develop simple and economic methods for evaluating strand-specific hybridization that uses the ease of homogeneous fluorescence assays with minimal DNA modification and enhanced signal amplification.

Conjugated polymers (CPs) are characterized by a delocalized electronic structure and can be used as highly responsive optical reporters for chemical and biological targets (10, 11). Because the effective conjugation length is substantially shorter than the number of repeat units, the backbone serves to hold a series of conjugated segments in close proximity. Thus, CPs are efficient for light harvesting and enable optical amplification via Förster transfer (12). Water-soluble CPs show exceptional fluorescence quenching efficiencies in the presence of oppositely charged acceptors and are of particular interest for transduction of biological recognition events (13).

Answer 2

The most common technique for detecting a specific DNA sequence that comprises a given biotech product is the polymerase chain reaction (PCR). This technique can be qualitative to indicate the presence or absence of a sequence or quantitative to determine the amount of DNA from a biotechnology-derived crop present in a sample. “Event-specific PCR methods” are designed to detect DNA sequences unique to a specific biotechnology-derived product. Alternatively, methods that detect DNA sequences that are common to many events are referred to as “screening methods”.