DNA ladder
A DNA ladder is a mixture of DNA strands of known different lengths that is used as a comigration standard (size standard, size marker) in agarose gel electrophoresis to determine the size of DNA in a sample . From the known amount of DNA in the comigration standard, in addition to the length, an estimated quantification of the DNA in a sample can be achieved. Furthermore, the term DNA ladder is used for a characteristic length pattern of an agarose gel electrophoresis of apoptotic cells.
A standard DNA ladder contains fragments between about 100 base pairs and 10,000 base pairs. Special DNA ladders can focus on particularly long or particularly short DNA fragments and thus facilitate a more precise determination of the size. Similarly, there are also ladders of a defined size made from single- and double-stranded RNA .
example
An example of a DNA ladder is shown in the figure on the right. It is a photo of a typical agarose gel (1% agarose) in UV light . The light bands are the DNA fragments that fluoresce red when stained with ethidium bromide in UV light. The DNA ladder is in the first lane, the sizes (bp for base pairs ) of the respective fragments are indicated on the side. The total amount of DNA of the applied ladder is 1 µg. DNA samples were separated in the second and third lane. The first sample contains four different sized fragments of about 4500, 2500, 1500 and 1300 bp. The second sample contains an 8000 and one 2500 bp fragment. The more intense bands show that the first sample contains slightly more DNA than the second sample.
Molecular weight determination
The mathematical relationship between the number of base pairs (approximately also the molar mass) and the distance covered in the agarose gel is logarithmic . The correlation can be linearized by means of a semi-logarithmic representation in a graph .
DNA ladder related to apoptosis
In connection with programmed cell death, the DNA ladder is used as a highly sensitive indicator of apoptosis . The phenomenon was first described in 1980 by Andrew H. Wyllie of the University of Edinburgh Medical School.
In the course of apoptosis, endonucleases cleave genomic DNA between the nucleosomes ( linker region ) and produce DNA fragments with a length of around 180 base pairs. The DNA ladder comes about because the DNA in the area of the nucleosomes is protected from degradation by the DNase , whereas the intermediate areas can be hydrolyzed. In necrosis, on the other hand, fragments of random sizes occur during DNA breakdown, which appear as "smears" in an agarose gel. The representation of the "apotoseleiter" is therefore a sensitive method to distinguish apoptosis from ischemic or toxic cell death.
literature
- Friedrich Lottspeich , Joachim W. Engels (Ed.): Bioanalytik . 2nd edition, Spektrum Akademischer Verlag, Heidelberg 2006, ISBN 978-3827415202 .
- Cornel Mülhardt: The Experimenter: Molecular Biology / Genomics. Sixth edition. Spectrum Akademischer Verlag, Heidelberg 2008. ISBN 3-8274-2036-9 .
Individual evidence
- ↑ Wyllie AH: Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation . In: Nature . 284, No. 5756, 1980 Apr 10, ISSN 0028-0836 , pp. 555-556. doi : 10.1038 / 284555a0 . PMID 6245367 .