Methods and technologies for DNA analysis

DNA analysis is the term used to describe a molecular biology procedure, the core of which is the examination of deoxyribonucleic acid (DNA). This macromolecule, which is characterized by its double helix and is present in every cell nucleus, is the carrier of the genetic material and consists of the main bases or nucleotides adenine, guanine, cytosine, thymine and uracil. These nucleotides are each arranged in base pairs in the double helix. Two bases are thus connected to each other with the help of hydrogen bonds. DNA analysis examines the specific arrangement of these base pairs. This arrangement is unique to each individual. Here you can learn more about the different methods and technologies for DNA analysis.

The development of DNA analysis

DNA analysis is used today for many different purposes. These include, first and foremost:

• The medical diagnostics
• The forensics
• The genealogy
• The genetics

Modern DNA analysis has its basis in the complete decoding of the human genome. This human genome project was started in 1990. All three million base pairs that make up human genetic information have now been completely decoded. With new technologies for sequencing and genotyping, DNA analysis is now much faster and more efficient than in the past.

How does DNA analysis work?

Several technological methods are available today for DNA analysis. The polymerase chain reaction in particular has significantly accelerated analysis in recent decades. Among the most important of these analytical methods are:

• The PCR
  The polymerase chain reaction, or PCR for short, was developed back in the 1980s. With their help, DNA sections can be copied in the laboratory as often as desired. This means that even small traces of DNA can be used for targeted analysis.
• Real-time PCR
  In this method, the individual DNA variations are analyzed using fluorescent dyes. The emission of these dyes occurs in real time, which has also given this method the name quantitative real-time PCR.
• DNA Microarray
  This technology simultaneously examines several thousand DNA sequences. These are located on the surface of the arrays or also called chips. When these arrays come into contact with a DNA sample, a fluorescent signal is released, which is measured photometrically and used for analysis.

The individual methods as well as alternative methods of DNA analysis are described in more detail below. Before DNA analysis is even possible, the DNA must first be extracted from cells.

From what is the DNA extracted?

DNA samples are usually blood, body fluids, but also hair or tooth enamel. The latter serves as a DNA source, especially in the case of burnt corpses, because the enamel is well embedded in the tooth and can therefore withstand high temperatures. Hair is often used for DNA extraction, but it must have a hair root. DNA is extracted from these samples in a specially approved laboratory. This is done with the help of various processes. For this, the cells must first be lysed. There are mechanical and non-mechanical methods for this dissolution of the cells. In mechanical dissolution, the cells are triturated with a pestle or crushed in special mills with the aid of tiny beads. In the chemical method, the cells are placed in a so-called buffer solution, which dissolves the cell walls.

The DNA extraction

After lysis, the DNA is finally extracted from the cell nucleus. In most cases, this is done by two-phase extraction or ethanol precipitation. Extraction is a process used to purify or separate liquid and solid substances between two immiscible solutions. Two-phase extraction takes advantage of the specific properties of biomolecules to distribute differently in aqueous solutions. Phenol-chloroform or phenol-chloroform-isomyl alcohol or trizol serve as solutions. In the process, RNA, DNA and proteins are separated from each other.

In a process known as precipitation, the DNA is separated by the addition of certain substances and sinks to the bottom of the test tube. An alcohol solution of ethyl alcohol or isopropanol is usually used for this purpose. Water has a different dielectric conductivity than ethanol. If ethanol is added to the sample, water is removed from the DNA, causing it to eventually sink to the bottom. The DNA becomes visible as a white, gel-like substance and can then be analyzed.

Specific methods of DNA analysis

Since the use of DNA analysis, several methods have developed for this purpose. PCR is one of the newest methods. Before that, DNA was analyzed using other technologies.

1. The fingerprinting
   This method of DNA analysis is one of the oldest and was first used successfully in criminalistics back in the 1980s. The DNA is cleaved with the help of specific enzymes. The resulting fracture sites differ in each individual, creating a typical genetic fingerprint. However, relatively large amounts of DNA are required for this form of DNA analysis. Usually 1 to 2 micrograms of DNA are needed for this. In addition, the DNA obtained must be of very high quality, i.e. high molecular weight.
2. The locus-specific polymorphisms
   This form of analysis is mainly used in the USA. Single-locus systems (SLS) are examined using the so-called RFPL method. By a single locus, scientists mean a specific position on a chromosome where a specific gene or marker is located. The abbreviation RFLP stands for restriction fragment length polymorphism. In the process, certain cutting patterns on the DNA strand can be identified. This method is mostly performed in conjunction with PCR, especially in medicine today.
3. PCR: Polymerase Chain Reaction
   With PCR, gene segments can be multiplied as often as desired. This is particularly advantageous when only small traces of DNA are available for analysis. PCR is used primarily in those cases where conventional methods of DNA analysis fail. However, PCR can also produce doubling patterns or other artificial by-products.

The technique of PCR

PCR is a relatively new method to analyze DNA. This analytical technique was developed by biochemist Kary Mullis in 1983. In 1993, he received the Nobel Prize for this work. The method is carried out in three steps.

1. Denaturation
   In this phase, the DNA is heated to around 95ºC. The heat dissolves the hydrogen bond between the strands, resulting in two single strands.
2. Hybridization
   In this step, the DNA is cooled down to 50º C again. In this process, the so-called DNA primers bind to the DNA strands and thus form the starting point for the complete PCR.
3. Elongation
   In this step, the DNA is finally copied. The sample is heated to 72ºC again. The result is an exact copy of the original DNA strand. This process is repeated as often as desired until a sufficiently large number of DNA strands are present.

What is DNA analysis used for?

Today, DNA analysis is mainly used in forensics. In this process, traces of blood or body fluids at crime scenes can be analyzed and assigned to a specific person. For the assignment of an unknown DNA, there are now special databases in which the genetic profile of criminals is stored. In Germany, for example, this is the DAD database at the Federal Criminal Police Office (BKA).
In addition, these analytical methods are also used in medical research to investigate diseases. Bacteria or viruses can also be identified with DNA analysis. In addition, DNA analysis is also possible for plants and animals. Furthermore, it is used today in the food industry for the analysis of plant or animal products.