How we get from sample to result in genomics

Biological research is becoming faster and faster through new methods. From the specimen to the genetic code – is everything a question of time?

In science, the calculation of time generally starts with the specimen to be investigated. If necessary, the specimen is stabilised. This method is recommended especially for the analysis of gDNA, ccfDNA, RNA, CTCs, or proteins from blood samples.

Every experiment starts with a specimen

First, specimen processing begins. In most cases, independent of the starting material (blood, tissue, virus, bacterium or urine), a nucleic acid extraction is performed. Conventional extraction kits, special extraction methods or automated systems can be used for this.

For anyone who is pressed for time, there are already master mixes available nowadays which contain polymerase which is so robust that direct amplification from the specimen without any purification at all is possible. Even difficult tissue, such as mouse ear clips, certain plants or yeasts can be directly analysed in a very time-efficient manner.

In diagnostics, following purification, the analysis is frequently conducted directly in the form of an amplification. This method of PCR is a very fast and cost-efficient variant, in particular for the detection of hereditary diseases or viral infections. In science, by contrast, a specimen is often further processed. For the cloning of genes, PCR is generally needed only for the amplification of the DNA fragment. Once the desired amount is available, modifying, cutting, and alloying are happily performed. Depending on the application, industry has the suitable products ready. From standard polymerases for “simple” amplifications to HiFi and mega HiFi polymerase, science can nowadays process nearly everything up to 20 kb.

Quality and quantity

Whoever wants to know not only whether a special nucleic acid is present but also how much of it is present uses the quantitative PCR method – known in short as qPCR. In this method, DNA dyes are used to determine the quantity of PCR products. However, to the chagrin of science, this detects specific as well as non-specific PCR products. For applications such as pathway analyses, microRNA detections, or multiplexing, specific fluorogenic probes are therefore often used for a target gene.

An amplified specimen should finally always be analysed on a gel. Only in this way is it possible to obtain information about the purity and the condition (degradation) of the specimen. Fortunately, most laboratories have nowadays moved away from the use of ethidium bromide and instead use so-called “safe dyes” which are less carcinogenic. Particular safe dyes are even already able to stain DNA and RNA separately from each other and thus provide a statement on DNA contamination in RNA specimens.

DNA molecule analysis

The sequencing of DNA as a final analysis is indispensable today. Here the purity of the specimen is particularly important; it is therefore recommended to perform purification before starting. There are special kits which remove enzymatic remnants from previous PCRs. Dye terminators from previous steps can also be easily removed with appropriate kits.

We can analyse the function and structure of entire genomes within a few hours and perform sequencing overnight. In comparison to this, conducting a typical amplification protocol with 30 cycles still took approximately 3 hours just ten years ago. Thanks to increasingly better reagents and kits, we can nowadays assume that the diagnosis, prognosis and treatment of cancer and many other illnesses will progress even faster in the next few years and we can use our valuable time even more efficiently. After all, time is relative!