Contaminant removal of non-pollen material in palynologic samples for DNA barcoding Pubblico

Abstract

DNA barcoding is a recently established technique that uses genetic markers to identify plant, fungal, and animal species. DNA barcoding for pollen, specifically, uses these genetic markers to identify pollen species. Barcoding will circumvent identifying pollen through visual microscopy, which will allow for rapid species-specific identification. This has broad implications since pollen is a biomarker of space and time and has functional importance in areas such as human respiratory allergies and forensic science. In certain circumstances, however, pollen samples may become contaminated with non-pollen plant material, which will not allow pollen species to be identified through the use of barcoding. This paper presents a series of trials conducted to alleviate this issue through the removal of non-pollen plant material with chemical and enzymatic reagents. Several combinations of chemical and enzyme treatments were tested on pollen and non-pollen material with the goal of removing all non-pollen DNA while maintaining the integrity of the pollen DNA. I found pre-treatment with sodium hydroxide, followed by treatment with DNase I enzyme was the best combination in achieving this goal. While this protocol showed promise, it was not consistently effective in removing non-pollen DNA. Using the foundations of this project, contaminant removal in a DNA barcoding context will require further methods development. Improving these methodologies in contaminant removal will allow for a broader range of powerful applications for DNA barcoding and pollen identification.

Table of Contents

INTRODUCTION 1

MATERIALS AND METHODS 5

Overview 5

Pollen collection 5

Chemicals used 6

Preliminary chemical treatment trials 6

Primary chemical treatment trials 7

DNA isolation 8

DNA amplification, visualization 8

DNA searches using Geneious 9

Data analysis 9

RESULTS 11

Overview 11

Preliminary results 11

Primary results 13

DISCUSSION 11

Overview 15

Preliminary trials 15

Primary trials 16

Caveats and future directions 17

Conclusion 19

REFERENCES 20

TABLES

Table 1 Pollen only (preliminary) 12

Table 2 Leaf only (preliminary) 12

Table 3 Anther only (preliminary) 13

Table 4 Ordinal logistic regression (preliminary) 13

Table 5 Binomial GLM on Leaf/pollen 14

Table 6 Linear model for Pollen only 14

Table 7 Linear model for Leaf/pollen 14

Table 8 Ordinal logistic regression (primary) 14

About this Honors Thesis

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School	Emory College
Department	Environmental Studies
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biology, Genetics Environmental Sciences
Parola chiave	pollen DNA barcoding
Committee Chair / Thesis Advisor	Brosi, Berry J, Emory University
Committee Members	Saikawa, Eri , Emory University Morran, Levi T, Emory University

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