Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA

• Published in: Molecular biotechnology Vol. 60; no. 2; pp. 124 - 133
• Main Authors: Machnik, Grzegorz, Skudrzyk, Estera, Bułdak, Łukasz, Ruczyński, Jarosław, Kozłowska, Agnieszka, Mucha, Piotr, Rekowski, Piotr, Szkróbka, Witold, Basiak, Marcin, Bołdys, Aleksandra, Sławska, Helena, Okopień, Bogusław
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.02.2018; Springer Nature B.V
• Subjects: Alleles; Astrocytes; Biochemistry; Biological Techniques; Biotechnology; Cell Biology; Cell lines; Chemistry; Chemistry and Materials Science; Correlation analysis; Deoxyribonucleic acid; DNA; Gene sequencing; Genetics; Human Genetics; Multiple sclerosis; Nucleic acids; Nucleotide sequence; Original Paper; Peptide nucleic acids; Placenta; Polymerase chain reaction; Protein Science; Reaction products; Transcription; Peptide nucleic acid (PNA); Strand invasion; Human endogenous retroviruses; Quantitative methods; Multiple sclerosis-associated retrovirus (MSRV); Polymerase chain reaction (PCR)
• DOI: 10.1007/s12033-017-0057-0
• ISSN: 1073-6085

In the presented assay, we elaborated a method for distinguishing sequences that are genetically closely related to each other. This is particularly important in a situation where a fine balance of the allele abundance is a point of research interest. We developed a peptide nucleic acid (PNA) strand invasion technique for the differentiation between multiple sclerosis-associated retrovirus (MSRV) and ERVWE1 sequences, both molecularly similar, belonging to the human endogenous retrovirus HERV-W family. We have found that this method may support the PCR technique in screening for minor alleles which, in certain conditions, may be undetected by the standard PCR technique. We performed the analysis of different ERVWE1 and MSRV template mixtures ranging from 0 to 100% of ERVWE1 in the studied samples, finding the linear correlation between template composition and signal intensity of final reaction products. Using the PNA strand invasion assay, we were able to estimate the relative ERVWE1 expression level in human specimens such as U-87 MG, normal human astrocytes cell lines and placental tissue. The results remained in concordance with those obtained by semi-quantitative or quantitative PCR.