Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Improvement, Standardization, and Validation of A Novel Genomic DNA Extraction Method for Human Breastmilk

Mario Iván Alemán-Duarte
ORCID logo ,
Blanca Rosa Aguilar-Uscanga
,
Guadalupe García-Robles
,
Felipe de Jesús Ramírez-Salazar
,
Israel Benítez-García
ORCID logo ,
Edgar Balcázar-López
* ,
Josué Raymundo Solís-Pacheco
*
Version 1 : Received: 13 October 2022 / Approved: 14 October 2022 / Online: 14 October 2022 (03:19:46 CEST)

How to cite: Alemán-Duarte, M.I.; Aguilar-Uscanga, B.R.; García-Robles, G.; Ramírez-Salazar, F.D.J.; Benítez-García, I.; Balcázar-López, E.; Solís-Pacheco, J.R. Improvement, Standardization, and Validation of A Novel Genomic DNA Extraction Method for Human Breastmilk. Preprints 2022, 2022100199. https://doi.org/10.20944/preprints202210.0199.v1 Alemán-Duarte, M.I.; Aguilar-Uscanga, B.R.; García-Robles, G.; Ramírez-Salazar, F.D.J.; Benítez-García, I.; Balcázar-López, E.; Solís-Pacheco, J.R. Improvement, Standardization, and Validation of A Novel Genomic DNA Extraction Method for Human Breastmilk. Preprints 2022, 2022100199. https://doi.org/10.20944/preprints202210.0199.v1

Abstract

Recent advances in DNA sequencing technology have shown that the human milk microbiota of healthy women varies substantially. The gDNA extraction method may influence the observed variation, biasing the microbiological reconstruction after all. In this study, a genomic DNA extraction method for DNA isolation from human milk samples was standardized and compared with commercial and standard hose make methods. Spectrophotometric measurements, gel electrophoresis, and PCR amplifications were used as criteria for evaluating the quantity, quality, and functionality of the extracted DNA. Furthermore, the standardized method of extracting gDNA from human milk was evaluated for its ability to isolate functional DNA from gram-positive, and gram-negative bacteria and fungi, to improve the reconstruction of microbiological profiles. The novel DNA extraction method increased the quantity and quality of the gDNA extracted compared with commercial and standard house-make protocols. This method even allowed PCR amplification of the V3-V4 regions of the 16S ribosomal gene in all samples, and the ITS-1 region of the fungal 18S ribosomal gene in 95 % of the samples as well. It is concluded that the proposed method provides better performance for the extraction of gDNA from complex samples such as human milk.

Keywords

human milk; DNA extraction; PCR; DNA quality, microbiota

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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