Electrophoretic Techniques for the Detection of Human Microsatellite D19S884

Background: The detection and analysis of microsatellites is very important for the mapping of genetic diseases because they are commonly used as genetic markers. Microsatellite marker D19S884 has been associated with polycystic ovary syndrome (PCOS), the most common reproductive endocrine disease of women in their childbearing years. It is responsible for an estimated 70% of cases of anovulatory infertility. In this work, we detected microsatellites in DNA extracted from the blood of PCOS patients.
Methods: DNA microsatellites were amplified by polymerase chain reaction (PCR) using a pair of specific primers tagged with fluorescence to yield products of 160–200 base pairs in length. Alleles were separated on 4% low-melting agarose gels; stained with a safe gel staining, GelRed™, which is an alternative to ethidium bromide; and visualised by ultraviolet illumination.
Results: Bands were observed, but their base-pairs differences were difficult to distinguish. To identify each allele clearly, the PCR products were also analysed using capillary gel electrophoresis for fragment analysis where it was possible to discriminate even in case of difference between two pairs of bases between the alleles.
Conclusion: In this article, we present a protocol that combines the use of gel electrophoresis and fragment analysis in the identification of genetic biomarkers for PCOS.

Keywords
microsatellite; D19S884; low-melting agarose gel; FBN₃; GelRed; fragment analysis