PCR and Gel Electrophoresis
PCR - DNA in abundance
Due to Covid-19, everyone should be familiar with the term PCR test by now. With its help, an infection can be detected. But how does it actually work? Stay tuned and find out why the PCR has not only been a breakthrough for diagnostic but also for genetic engineering in the last decades.
The PCR is a brilliant method to copy specific parts of DNA sequences. The method is orientated towards the natural process of DNA replication, which plays a major role in cell division. It ensures that every daughter cell contains exactly one copy of the cell’s genetic information. Taking this process as an example, an artificial concept was born to amplify DNA sequences – called PCR or Polymerase Chain Reaction. But which steps does a PCR include? And how can it be explained on a molecular level?
DNA consists of two complementary strands, built from base pairs. These base pairs being complementary means that every base is only binding to exactly one partner in the opposing strand. Adenin (A) is only binding to Thymine (T), Cytosine (C) is only interacting with Guanin (G). This being said, the first step of replication, but also of the artificial PCR, includes the separation of this DNA double-strand. Both of the forming single strands code for the same genetic information, which is why they can be used as templates for new DNA molecules. The separation is performed by heating the DNA sample, which breaks the bonds between the double strand’s base pairs.
For the second step of the process, it is necessary to target a specific sequence in the DNA, which shall be copied. This is achieved by so-called primers. Primers are single-stranded, short DNA fragments that bind to the part in the DNA which shall be copied. This marks a starting point for a certain enzyme: the DNA polymerase. This enzyme is able to fill up the complementary bases and, therefore, creates two new double strands – on the template of two single strands. Therefore, an exponential duplication is achieved – meaning that with every cycle, the DNA strands are doubled.
But how is this method connected to the detection of Covid-19?. Covid tests are based on the detection of viral genetic information. If viral genes are present in a sample, we use a specific primer for the amplification. In the Covid PCR every amplification of the viral genes gives a signal. The higher the viral load, the stronger the signal.
However, PCR is not only of particular importance for diagnostic – but also for every step performed in genetic engineering. PCR offers the possibility to amplify and modify even small amounts of DNA sequences and, therefore, contribute to the genetic modification of several organisms.
Gel electrophoresis and its importance for genetic engineering
If you’re interested in thrillers and crime stories, we will give you some eye-opening facts today. Have you ever wondered how the police convict a criminal – only with small parts of fingernails or hair? Thanks to everyone’s unique genetic fingerprint, perpetrators can be identified easily. But what defines your genetic fingerprint and how can it be compared between different individuals?
The technique you use for this analysis is called gel electrophoresis. With its help, protein chains or, in our case, DNA sequences can be separated by their individual size and charge. For the determination of the genetic fingerprint, repetitive regions in your genome are compared – the so-called simple sequence repeats (SSRs). The repeats are located in the exact same region in your genome, they only differ in the number of repeats and, therefore, in their length.
Now, gel electrophoresis comes into play. Because it is the main technique to compare the length of DNA sequences. The technique is based on a pore forming gel, similar to a net, to which voltage is applied. Due to the negative charge of DNA, the fragments migrate to the positively charged pole. Large DNA fragments are not able to move fast in the gel – the small pores hold them back. Precisely the opposite case is true for short DNA sequences – they are much more agile and, therefore, migrate further than long fragments. This is why the comparison of the unique repeat length by electrophoresis is a certain way to convict a perpetrator by its genetic fingerprint.
However, gel electrophoresis is not only used for fingerprint analysis, but it is one of the most common methods in the biochemistry lab. Every step of genetic modification can be verified by measuring the size of certain DNA fragments – for example of a specific gene you wanted to transfer into your host organism. Amplifying the genetic sequence through PCR and comparing the sequence length to a standard marker in gel electrophoresis, gives information about the success of your experiment. This way, you can also isolate genes for further analysis or search for proteins with the right size in your sample.