Date of Award

Spring 5-2020

Document Type

Thesis

College

College of Arts and Sciences

Degree Program

Human Nutrition

Degree Name

Master of Science

Thesis Advisor

Dr. Hope Lima

Committee Member

Dr. Matthew Stern

Committee Member

Dr. Katherine Knop

Keywords

Human milk, Breastfeeding, Lactation, DNA extraction, DNA optimization, Quantitative polymerase chain reaction (qPCR)

Committee Member

Dr. Duha Hamed

Abstract

Background: Techniques are emerging for determining the best and most cost-effective way to test for human milk adulteration. Currently, the literature is focused on the use of qPCR testing, a technique used to isolate and amplify pieces of DNA for analysis. However, no recommendation currently exists on the best DNA extraction kit to use to achieve optimal DNA yield or purity from human milk samples for downstream qPCR use. Thus, the objective of this study is to assess and compare two DNA extraction kits for use with human milk samples for future DNA-based analysis in the testing for bovine milk adulteration in human milk.

Methods: Forty mothers pumped human milk samples under the observation of a researcher using a brand-new hand pump. Eight unadulterated samples were then randomly chosen for DNA-extraction. The eight samples were thawed, pooled, and DNA was isolated using the Omega Bio-Tek's E.Z.N.A.® Blood DNA Mini Spin Kit and the Norgen Biotek Corporation Plasma/Serum Circulating DNA Purification Mini Kit (Slurry Format) per the protocols included in the kits on arrival. An overnight incubation modification was also added to both kits to try to obtain optimal yield and purity. UV/VIS spectroscopy was used to determine DNA yield and purity using the ThermoFisher Scientific NanoDrop 2000TM 260/280 ratio, and a cost comparison was done between kits.

Results: The Norgen kit with no modification provided 143% more DNA than the E.Z.N.A kit with no modifications. Similarly, the average nucleic acid yield was 134% greater when comparing the Norgen and E.Z.N.A kit with an overnight incubation. The Norgen kit provided a 17.0% greater 260/280 ratio and an 11.4% greater 260/280 ratio than the iii E.Z.N.A. kit, with and without modifications, respectively. The Norgen kit costs $2.37 more per extraction than the E.Z.N.A kit. Modifying both DNA extraction kits with an overnight incubation decreased the average nucleic acid yield and purity of the resulting DNA.

Conclusion: From these results, the Norgen kit without overnight incubation is a better extraction kit for DNA extraction from raw human milk for both nucleic acid yield and purity. However, the EZNA kit costs less per extraction at $1.45 vs. $3.82. For extraction purposes, purity should be prioritized over nucleic acid yield because contaminants can compromise results and shorten the shelf-life of samples.

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