Author/Editor		Zrimec, Jan; Kopinč, Rok; Rijavec, Tomaž; Zrimec, Tatjana; Lapanje, Aleš
Title		Band smearing of PCR amplified bacterial 16S rRNA genes
Type		članek
Vol. and No.		Letnik 95, št. 2
Publication year		2013
Volume		str. 186-194
ISSN		0167-7012 - Journal of microbiological methods
Language		eng
Abstract		Band smearing in agarose gels of PCR amplified bacterial 16S rRNA genes is understood to comprise amplicons of varying sizes arising from PCR errors, and requires elimination. We consider that with amplified heterogeneous DNA, delayed electro-migration is caused not by PCR errors but by dsDNA structures that arise from imperfect strand pairing. The extent of band smearing was found to be proportional to the sequence heterogeneity in 16S rRNA variable regions. Denaturing alkaline gels showed that all amplified DNA was of the correct size. A novel bioinformatic approach was used to reveal that band smearing occurred due to imperfectly paired strands of the amplified DNA. Since the smear is a structural fraction of the correct size PCR product, it carries important information on richness and diversity of the target DNA. For accurate analysis, the origin of the smear must first be identified before it is eliminated by examining the amplified DNA in denaturing alkaline gels.
Keywords		bacterial diversity metagenomics bioinformatics bakterijska raznolikost metagenomika bioinformatika