zum Inhalt springen

DNA-Sequencing

DNA-Sequencing is a method to determine the sequence of a DNA molecule, which was developed by Frederick Sanger in 1977.

It requires a small piece of know sequence in the DNA molecule of interest to which the primer binds and from which the sequencing starts. If the entire DNA molecules is unknown, it can be pasted into another known DNA molecule that can be propagated in bacteria (i.e. DNA cloning). The steps of a sequencing reaction is similar to a PCR-reaction (denaturation, annealing, extension), but with just one primer, which results in a linear instead of an exponential amplification. In addition to the standard four nucleotides A, T, G and C, four modified nucleotides (dideoxy-nucleotides) are added in low concentrations, which will prohibit the further the extension of the newly synthesized DNA molecule, when incorporated. Because the incorporation of the modified nucleotides is at random, many DNA molecules of all sizes ranging from few base pairs to hundreds of base pairs in length will be generated. Consequently, the modified nucleotide is always at the end of each of the DNA molecules. Each of the modified nucleotides is labeled with a unique color (fluorophore), which can be detected by its fluorescence. Because all fragments of a specific length have the same labeled nucleotide at their end, the base at this specific position can be determined by its color. To determine the sequence of the DNA, the sequencing reaction is electrophoretically separated. The small fragments will reach the place where the fluorescence is measured first, the largest fragments last. Each fragment of a specific length will yield in a fluorescence peak, which tells what base is at the respective position of the sequenced DNA molecule.

Next Generation Sequencing – NGS

NGS is a method that allows the sequencing of millions of DNA templates in parallel in a cost-efficient and fast manner.

Because many different NGS protocols from different companies are around and due to the complexity of the sample preparation and sequencing process, only one widely used NGS system (Illumina®) is explained here.

The DNA of interest is first chopped in to smaller pieces of a relatively uniformed size and are ligated with adaptors to which the primers will bind. These fragments (the library) are attached to a solid carrier for example a glass slide. The billons of different DNA fragments are then clonally amplified on the carrier. Sequencing is performed in real-time by adding a DNA polymerase and reversibly fluorescently labeled nucleotides. For each base of the millions of different templates that is to be sequenced, the polymerase will add the respective nucleotide. Then the whole glass slide is excited by laser light, which will lead to a fluorescent signal for each newly added base of each fragment (as for Sanger sequencing, the four bases again have four different colors) and a picture is taken. Then the label is removed and the process is repeated many times. The resulting reads are between 50 and 250 base pairs. The total amount of sequence is around some billions of base pairs, which are highly redundant. The reads are assembled usually using a reference sequence to which they are mapped.

Exome sequencing

Exome sequencing is a method to determine the sequence of all protein coding regions of a genome by means of next generation sequencing (NGS).

Eukaryotic genes have a modular structure. Short DNA stretches that encode the amino acid sequence of the respective protein are separated by non-coding regions call introns. Introns are variable in length and can be very long, whereas exons are usually short and are around 250 base pairs in length.

The actual protein coding information of all exons of the human genome (3 x 109 base pairs) is only about 1% and is called exome. However, the majority of all disease causing mutations are located in exons. Because to total size of the exome is so much smaller that the genome itself it is possible to drastically reduce the sequencing costs, the speed or to increase the coverage of a given sequencing run, when only the exome is analyzed.

Exome sequencing requires efficient selection and enrichment of exons. Several strategies have been described so far. Exemplarily, one possible experimental is briefly described in the following.

Isolated genomic DNA is fragmented and the fragments are size selected. After ligation of adaptor sequences to both ends, the fragment are then hybridized to a microarray containing probes specific to all known exons. For the human genome this represent about 180.000 different probes. After hybridizing the genomic fragments to the micro array and subsequent washing, only those fragments that contain a sufficient piece of exonic sequence remain bound to the micro array. This fragments can be isolated, amplified by conventional PCR via the previously ligated adaptors and then subjected to next generation sequencing (see NGS), which allows determination of the sequence and assembly of the exome of the specimen/patient.

RNA-Seq

RNA-Seq ist used to analyze the whole transcriptome of a sample (e. g. cells, tissue etc.) by next generation sequencing (NGS).

The transcriptome is the sum of all expressed RNAs of a sample, which can be coding (mRNAs) and non-coding. To analyze the transcriptome of a given biological sample. the RNA is isolated and transcribed into cDNA. The cDNA library is then analyzed by NGS and the resulting sequences are assembled and mapped to a reference genome.

Fluorescence activated cell sorting – FACS

Is a method to isolate individual cells based on optical features of the cells.

The physical background of each of the steps of the method is rather complicated and won’t be explained here. Briefly, the main steps are hydrodynamic focusing of the cells leading to a stream of fluid in which the cells are aligned one after the other, controlled break up the flow of the fluid into droplets containing the cells, passing the droplets through several laser beams, detecting the emitted signals in real time and sorting the droplets according to this information. The drops are sorted into different collection vessels by applying an electrical field to them, which allows to control the direction of movement of each of the droplets.

Most of the times the cells are stained with antibodies (see IHC/ICC/IF) and then sorted according to the fluorescence of the antibodies. Often antibodies that recognize antigens on the cell’s surface are used, allowing staining and sorting of living cells. Obviously, living cells are much more versatile regarding downstream applications compared to fixed (dead) cells.