Fluorescence in PCR Diagnostics
PCR diagnostics makes it possible to detect and sequence DNA. RT-PCR is used to detect or sequence RNA. In a first step, the RNA is transkripted into DNA using reverse transkritase and then amplified in the polymerase chain reaction (PCR). Finally, the DNA sections are sequenced.
By Dr. Jakob Bierwagen, AHF analysentechnik AG
Base labeling with fluorescent markers
A wide range of methods are available for sequencing. The individual bases are usually labeled with fluorescent markers. The classical method according to Frederick Sanger (1918–2013, Nobel Prize 1980) works in such a way that the complementary strand is extended by the PCR starting from a (short) known section (primer). As in ‘normal’ amplification, the DNA double helix is separated by heating. The single strands are now separated into four polymerization experiements. A different base is fluorescence-labeled in each of the four approaches. In addition, a small amount of dideoxynucleotide triphosphate (ddNTP), a double deoxygenated base, is added to each of the four approaches. When incorporated during the polymerase chain reaction, this non-natural DNA base leads to a termination of the chain reaction, since it does not contain an OH group in its 3'-position. Therefore, if this base is incorporated during the PCR, the chain reaction for this molecule ends at this position. This results in fragments of different lengths of the complementary DNA strand, which breaks off at the same base (A, C, G or T) in each of the four approaches.
Separation of the DNA fragments with gel electrophoresis
After this sequencing reaction, which is carried out in the same way as the PCR, DNA fragments of different lengths are found in the four approaches, which are separated by gel electrophoresis according to length/weight. By comparing the four approaches (by visualizing the fluorescence) the (complementary) sequence can be read.
Separation with capillary electrophoresis
Today, the four different ddNTPs are labeled with different fluorescent dyes. This makes it possible to add all four ddNTPs in one reaction vessel and to avoid separation into the four different approaches. Instead of gel electrophoresis, separation is performed by capillary electrophoresis. The end of the capillare is positioned into (four) laser beams. The fragments of different weights are then excited one after the other and each fragment shows the corresponding base by fluorescence in a different color. The sequence of the color signals thus directly reflects the sequence of the bases.
Detection with fluorescence further relevant
In addition, there are now second and third generation sequencing methods that are highly complex and automated, some of which work on a single molecule basis, but still use fluorescence as a detection method.
Highly selective filters required
Highly selective optical filters (>OD6) are required for the detection of the color code by the fluorescent molecules. Since only smallest amounts are detected, they must also have a very good transmission of >95%.
AHF is happy to advise you in selecting the right optical filter for your specific requirements. You benefit from our wide product range of optical filters and our longterm experience in the field of fluorescence applications.