A DNA Tracer System for Hydrological Environment Investigations

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Critical Review

A DNA tracer system for hydrological environment investigations renkuan liao, Peiling Yang, Wenyong Wu, Dan Luo, and Dayong Yang Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 23, 2018

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Environmental Science & Technology

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A DNA tracer system for hydrological environment investigations

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Renkuan Liao1, 2, Peiling Yang3, Wenyong Wu1, Dan Luo4, 5, 6*, Dayong Yang2*

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1. State Key Laboratory of Simulation and Regulation of Water Cycles in River Basins,

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China Institute of Water Resources and Hydropower Research, Beijing 100048,

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P. R. China

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2. School of Chemical Engineering and Technology, Key Laboratory of Systems

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Bioengineering (Ministry of Education), Collaborative Innovation Center of Chemical

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Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, P. R. China

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3. College of Water Conservancy and Civil Engineering, China Agricultural

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University, Beijing100083, P. R. China

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4. Department of Biological & Environmental Engineering, Cornell University,

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Ithaca, New York 14853, United States

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5. Kavli Institute at Cornell for Nanoscale Science, Cornell University, Ithaca, New

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York 14853, United States

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6. CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and

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Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, P.R. China

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Corresponding authors: Prof. Dayong Yang ([email protected]); Prof. Dan

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Luo ([email protected])

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ACS Paragon Plus Environment


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Abstract

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To monitor and manage hydrological pollution effectively, tracing sources of

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pollutants is of great importance and also is in urgent need. A variety of tracers have

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been developed such as isotopes, silica, bromide and dyes, however practical

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limitations of these traditional tracers still exist such as lack of multiplexed,

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multipoint tracing and interference of background noise. To overcome these

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limitations, a new tracing system based on DNA nanomaterials, namely DNA tracer,

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has already been developed. DNA tracers possess remarkable advantages including

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sufficient species, specificity, environmental friendly, stable migration and high

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sensitivity as well as allowing for multi-points tracing. In this review article, we

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introduce the molecular design, synthesis, protection and signal readout strategies of

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DNA tracers, compare the advantages and disadvantages of DNA tracer with

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traditional tracers, and summarize the-state-of-art applications in hydrological

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environment investigations. In the end, we provide our perspective on the future

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development of DNA tracers.

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Keywords:

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environment investigations; Environmental protection

DNA

tracers;

Pollution

sources

identification;

2


Hydrological

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Introduction

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Environmental pollution is one of the grand global challenges, which imposes

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negative impacts on human health, social and economy sustainable development1–5 .

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Tracing the source and migration pathways of pollutants in water, soil and

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atmosphere is one of the most important prerequisites for solving environmental

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problems6–10. Tracing refers to the use of labeled substances to track a specific

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substance in the natural environment for its movement paths, distribution, metabolic

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processes, or patterns. Tracing technologies have been widely applied in biology,

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chemistry and agriculture

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environment investigations, numerous natural and artificial tracers have been

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developed to determine migration pathways in natural environments. These

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traditional tracers included natural silica, stable isotopes, artificial bromide,

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chloride, fluorescent and etc17–22. Unfortunately, the ubiquity of natural tracers in

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water bodies resulted in environmental background 23. On the other hand, although

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some artificial tracers could overcome environmental background, the introduced

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artificial chemicals were difficult to identify due to the dilution effect in the huge

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water bodies24,25. Migration properties and the degradation rate of artificial tracers

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in multi-points tracing were arduous to control26,27. In addition, some tracers such as

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fluorobenzoic acid were expensive for use or analysis28,29. Moreover, high

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concentrations of artificial tracers were needed in the water bodies to achieve an

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adequate accuracy, which subsequently had potential adverse effects on

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environment30,31.

11–16

. Specifically, for the application of hydrological

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Nanoscience and nanotechnology provided alternative tracing strategies to

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address these challenges in hydrological environment investigations 32–37. In particular,

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tracers based on DNA nanomaterials (termed as DNA tracers) possessed

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overwhelming advantages and have exhibited promising performance. For instance,

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DNA tracers could be easily distinguished from environmental background due to the

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unique DNA sequence of each tracer. Well-established molecular biology 3



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technologies such as polymerase chain reaction (PCR) were able to amplify DNA

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exponentially, theoretically rendering the ultimate detection sensitivity: identification

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of DNA down to one molecule. In addition, the synthesis and usage of DNA were

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simple, cost-effective and environmental friendly38–40. DNA tracers have been

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demonstrated effective in environmental protection41–46. Environmental sciences in

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general were dealing with much larger scales than those of nanoscience and

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nanotechnology. Consequently, the power of nanoscience and nanotechnology has

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not been fully adopted by environmental science yet. To this end, we organize this

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review in hope to bridge nanoscience and nanotechnology with environmental science.

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We systematically summarize the design, synthesis, protection and signal readout of

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DNA tracers, and discuss their applications in hydrological environment

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investigations. Furthermore, we provide our perspectives on the future directions of

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DNA tracers. A conceptual scheme is presented showing how DNA tracers work in

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identification of stream pollution sources (Figure 1).

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Synthesis of DNA tracers

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Property of DNA as tracer

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DNA is a well-known genetic molecule in living organisms. Until Seeman’s first

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demonstration of artificial DNA nanostuctures in 198247, the concept of using DNA

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beyond biology as a generic (more than a genetic) molecule was unheard of. To date,

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this alternative view of DNA has become more and more accepted with the synthesis

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of increasing number of DNA-based functional materials 48–58.

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In general, DNA can be single stranded (ssDNA) or double stranded (dsDNA).

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Each DNA strand was made of a phosphate-deoxyribose backbone plus four bases:

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adenine (A), guanine (G), cytosine (C) and thymine (T)59,60. Two complementary

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ssDNA strands were hybridized into a double helical structure by obeying the

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Watson–Crick base-pairing rule: A with T and C with G. In comparison with other

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synthetic materials, DNA possessed a variety of overwhelming advantages including

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its biological function, biocompatibility, molecular recognition capacity and

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nanoscale controllability.

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Compared with the limited number of species of traditional tracers, the number of

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DNA tracers (i.e., the combinatorial possibilities of AGCT four bases) was

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theoretically unlimited, sufficiently enough for almost any tracing usage. For example,

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a DNA strand with 100 base pairs (bp) can be designed with 4100=1.61×1060

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individual sequences61, and each sequence was differently specific which can be used

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as a unique tracer, ensuring adequate diversity and specificity of DNA tracers. This

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capability to produce unprecedented and unlimited individual DNA tracer allowed for

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multi-points tracing of different pollutant sources simultaneously. It was noteworthy

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that the lengths, diameters and volumes of DNA tracers could be rationally designed,

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and resultantly the physical/chemical properties could be adjusted, resulting in the

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precisely tuned migration activities. In particular, the similar chemical structures of

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different DNA tracers led to uniform degradation stability62. 5



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Regarding the cost for the fabrication of DNA tracers, it has been reported that

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the cost was 0.02 ¢/L of product by the encapsulation of silica46. It was less than $1

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per gram by the encapsulation of polylactic acid (PLA) microspheres, and the expense

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for reagent cost and analysis was approximately $2-$3 per sample63. Pang et al. only

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used 36 μg DNA tracer in the groundwater experiment, which was 7-orders of

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magnitude less than the quantity of Br tracer used (568 g). Likewise, compared with

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the amounts of chloride salt (CaCl2 5.7 kg) and rhodamine dye (32 g) used at the same

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site for similar tracing effects, the quantity of DNA needed in this study was about 8-,

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and 6-orders of magnitude less, respectively68. The details for advantages and

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disadvantages of the DNA tracer techniques in comparison with traditional tracers are

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listed in Table 1.

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Design principle

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To provide an overview, we sketch a flowchart for the fabrication of DNA tracer

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(Figure 2). In general, fabrication of DNA tracers includes four key steps: sequence

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design, specificity analysis, synthesis, protection. The quality of water played

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important roles in pollutant tracing such as affecting DNA degradation and migration,

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which essentially determined the fabrication strategy of DNA tracers. Water body was

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categorized into two types according to the quality: 1) clean water with low

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concentration of catabolic enzymes, neutral pH levels, and weak microorganism

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activity; 2) complex water with high concentration of catabolic enzymes, non-neutral

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pH levels and strong microorganism activity. In clean water, DNA tracers could be

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used directly. In contrast, in complex water, protection of DNA tracers was highly

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necessary prior to usage.

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Regarding the design of DNA tracers, three important steps should be taken into

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consideration: ssDNA or dsDNA, the DNA length and DNA sequences. At present,

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most DNA tracers were ssDNA. In most of studies, DNA were synthesized through

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chemical methods and were convenient to obtain from commercial vendors64–66. On 6


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the other hand, biosynthesis such as PCR generally produced dsDNA with high

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efficiency39. The length of the most commonly used DNA tracers ranged from 70 to

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500 bases. The critical consideration of DNA sequence was the avoidance of

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background from environmental DNA of organisms. In other words, the sequences of

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DNA tracer should be distinguished from genetic background DNA existing in

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environment.

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（http://www.ncbi.nlm.nih.gov/）could be used to check the designed DNA, ensuring

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the specificity63.

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Protection

A

readily

available,

public

domain

software

BLAST

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In real-world scenarios, the water condition was extremely complex such as

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containing a high concentration of catabolic enzymes or hazard chemicals, having a

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non-neutral pH value or high temperature, being exposed to the sunlight, and

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constituting of strong microorganism activity. If without any protection, these

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complex conditions reduced the stability of the DNA tracers, leading to low recovery

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rates and unprecise detection. Furthermore, DNA molecules tended to adsorb to

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sediments, soils and aquifer media, thus reducing the recovery rate of DNA. For

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example, Sabir et al. reported that the main reasons for tracer loss in the porous media

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were microbial degradation and sorption, and the mass loss of small DNA molecules

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was due to more frequent collisions with the porous material42. Foppen et al. found

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that the recovery rate of DNA tracers decreased from 55% at 112 m to 19% at 1192 m

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downstream of injection point (as a comparison, NaCl tracer varied only from 112%

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to 94%); they attributed this reduction of DNA tracers to the adsorption, attachment,

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decay and biological uptake in the stream67. Foppen et al. further explored the cause

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of DNA attenuation and quantified the attachment rate coefficients of DNA tracer on

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sediment and fitted the advective and dispersive transport parameters. They pointed

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out that the mass loss was due to both initial losses and attachment or sorption of

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DNA tracer in storage zone64. In a recent study, Pang et al. evaluated the attenuation

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of DNA tracer both in column and groundwater field tracing experiments, the 7



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adsorption to the aquifer media and its degradation were considered the main cause

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for the mass loss of DNA68.

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On the other hand, many studies indicated that DNA was apt to absorb onto the

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surfaces of clay, silica, polymer or humus and thus DNA tracers could be protected by

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these materials69–73. Therefore, the protection materials for DNA tracers could be

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selected from those materials. In particular, Yang and co-workers demonstrated that

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DNA was preserved in a clay mineral based hydrogel74. More than 90% DNA

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absorbed on the clay hydrogel; consequently the clay hydrogel provided a protective

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environment for DNA against DNase (Figure 3). Similarly, Mahler et al. studied the

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behavior of mobile colloids and sediment in hydrology after encapsulating DNA in

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clay, and their results showed that short strands of synthesized DNA reversibly

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adsorbed to both Na-montmorillonite and powdered silica surfaces via a magnesium

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bridge39. Silica has also been used to protect DNA tracers. Foppen et al. fabricated

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DNA tracers with a magnetite core on which DNA molecules were adsorbed, and

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being covered with a silica layer65. Puddu et al. reported that DNA was adsorbed on

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magnetic support, and the surface was coated a dense silica layer with the

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DNA/magnet nanocluster as invisible tracing tags. Their results indicated that DNA

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tracer encapsulated within the magnetic particles was stable for 2 years in decalin at

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room temperature46. The similar protection method using silica was reported by Mora

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et al., in which they applied submicrometer sized silica particles with encapsulated

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DNA as a tagging agent to evaluate pesticide drift45. In addition, some polymers such

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as polylactic acid (PLA) were employed to encapsulate DNA for the protection.

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Sharma et al. used PLA microspheres as a protective carrier of DNA tracer and

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introduced paramagnetic iron oxide nanoparticles inside tracer; such design not only

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improved the protection of the DNA tracer, but also enhanced the enrichment of DNA

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tracer, thereby improving the accuracy of the downstream quantitative PCR

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detection63.

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Signal amplification and readout 8


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The most prominent advantage of DNA tracers was the availability of powerful

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molecular biology tools such as PCR which efficiently amplified signal readout. Most

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of DNA tracers relied on PCR-based detection approaches, especially the

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biotechnology of real-time fluorescence quantitative PCR (qPCR). A general protocol

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for qPCR is outlined in Figure 4. Two key design factors included the length and the

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melting temperature of primers, which were critical in achieving sufficient signal

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amplification. Some websites and software, such as https://www.idtdna.com/site and

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Gene Designer (ATUM, USA), provided useful tools for primer design and

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optimization of testing conditions. qPCR showed excellent sensitivity in detecting

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DNA tracer. For example, Foppen et al. reported a detection limit of 50–100

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molecules/well67, and in Aquilanti’s work they achieved 10 molecules/well43. qPCR

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depended on either non-specific fluorescent dyes75–77 or hybridization probes as signal

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readouts78,79. Non-specific fluorescent dyes such as SYBR Green represented a

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straightforward readout fashion, which were directly applied to any DNA tracer and

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did not require fluorescently labeled target-specific probes80,81. However, non-specific

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fluorescent dyes tended to bind with all DNA presented in the reaction mixture which

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might include non-specific PCR products and primers, causing potential background

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noise and false positive82,83. In contrast, hybridization probes only recognized target

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DNA, which as a result significantly increased specificity even in the complex

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water84,85. Although qPCR was a powerful tool, there still existed some inhibitors in

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complex environmental water samples which interfered with qPCR detection. As

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reported by Foppen et al., the serial dilution errors, random errors of qPCR apparatus,

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the preparation of the qPCR mix, limited range of standard curve and water quality

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might cause errors in detecting DNA traces64. In addition, the mutual interference of

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different DNA tracers in water samples, turbidity of water samples, pipetting errors

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were also considered to be influence factors to interfere with qPCR measurement44.

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The errors for data obtained from qPCR technique could be reduced by improving

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design of oligos and primers, however, the inhibitors caused by the experiment steps

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and properties of protection material seemed inevitable63. Therefore, more active

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strategies such as oligo and primer optimization, water sample purification, protection 9



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materials selection are encouraged to be explored in future to reduce inhibitors in

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environmental water samples on the qPCR measurement.

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Alternatively, detection of DNA in other better-developed fields such as clinical

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diagnostics has provided informative strategies for signal readouts beyond

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fluorescence. These methods could be adopted in hydrological environment

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investigations, such as resonant light scattering, electrochemistry and elemental

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analysis 86–90.

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Case studies

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Numerous studies have demonstrated the promising advantages of DNA tracers

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in hydrological environment investigations. We herein exhibit some important

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research works as case studies (Table 2). Mahler et al. introduced a DNA-labeled

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method for tracing particle transport by encapsulating DNA molecules, and

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demonstrated that DNA tracer could be detected and read by using PCR and DNA

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sequence analysis39. In a later report, Sabir and co-workers indicated that DNA tracers

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could be effective collected from microliter samples of water taken from multi-level

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samplers at various distances from the injection point and be identified by PCR

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analysis41. They further carried out a few field experiments in Norway, in which they

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compared DNA tracers with traditional tracers in different situations of groundwater,

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such as porous media and fractured hard rocks42. Their results indicated that DNA

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tracer was much more effective in tracing groundwater flow and discriminating

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sources of pollutants than conventional tracer.

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qPCR provided a robust tool for the qualitative analysis of DNA with

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unprecedented sensitivity, which became one of the most powerful components in the

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readout of DNA tracers. Ptak and co-workers first employed qPCR to qualitatively

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measure the concentration of recovered DNA tracer from groundwater, which

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accurately portrayed the flow patterns of water and pollutants62. They obtained

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quantitative breakthrough curves for DNA tracer by performing qPCR measurement

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at different scales, i.e., laboratory column (50 cm length, 10 cm diameter) and 10


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field-scale (10 m span). Harnessing the power of qPCR, DNA tracers were used to

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study more complex hydrogeological scenarios, such as surface water, torrents water,

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effluent discharges and hydraulic contact of aquifers that were sensitive to

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anthropogenic activities. Foppen et al. simultaneously utilized six DNA tracers to

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study the hydrology of surface water. Their results confirmed that DNA tracers

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performed effectively in tracing surface flow-paths, with an ultrasensitive

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measurement of DNA molecules64,67. Bovolin et al. conducted bedrock river

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experiment with DNA tracers in torrents water, and their results confirmed the

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effectiveness of artificial DNA in fast flowing environment91. Pang et al. applied

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DNA tracers in tracking the effluent discharges in soils and groundwater68. In addition,

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Aquilant and co-workers tested the transport characteristics of DNA tracers in a

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column experiment, which showed an almost pure convective flow of DNA43.

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Aquilant et al. further reported a field-scale experiment, in which DNA tracers were

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effectively employed to explore the hydraulic contact of aquifers66.

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During the movement with water flow, the amount of DNA tracer was

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substantially reduced due to adsorption, attachment, decay and biological uptake.

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Adequate mass recovery of DNA tracer was therefore an essential factor to ensure

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sufficient amount of DNA for measurement, which guaranteed the accuracy.

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Generally, the effective and straightforward solution to improve mass recovery of

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DNA tracer was to protect DNA from adsorption, attachment, decay and biological

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uptake. With the continuous improvement of protection methods, the recovery of

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DNA tracer was gradually increased. Sharma et al. proposed PLA microspheres to

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protect DNA molecules, and their results demonstrated the effectiveness of the

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protection63. Furthermore, Dahlke et al. encapsulated DNA tracers in PLA

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microspheres, and achieved 66% recovery44. In addition, Foppen et al. used the silica

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layer to prevent DNA from decay, and their results indicated that around 70% mass of

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the DNA was recovered65. Similarly, Grass et al. used silica particles to encapsulate

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DNA tracers, which indicated that more than 95% of silica particles were found in the

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sedimented sludge92. 11



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Summary and outlook

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In conclusion, in this review we demonstrate that DNA tracer is an effective

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nano-technological tool applied in hydrological environment investigations. We

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provide an overview of the design, construction, protection and readouts strategies of

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DNA tracers as well as the application cases. DNA tracers possess remarkable

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advantages in comparison with traditional tracers, including environmental

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friendliness, stable migration and high sensitivity as well as allowing for multi-points

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detections. Inevitably, as exogenous materials to environment, one of the concerns is

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environmental safety. DNA molecules are essential constituents of life, which have

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existed ubiquitously in food, air, water, and also in almost all life forms. Therefore, it

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is naturally nontoxic. In addition, the sequences of DNA tracers are all artificially

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designed and chemically produced, but not from the genome of any organisms;

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therefore they neither possess any hereditary properties nor carry any potential risk of

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introducing foreign genes to any hosts.

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So far the development of DNA tracers is still at the nascent stage and some

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challenges still remain. From the material perspective, challenges include scale-up of

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material manufacturing, further reduction of manufacturing cost, and improvement of

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protection and mass recovery. From the hydrological perspective, challenges include

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multi-points detection, application in large scale water body or severe water

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environment and development of migration simulation models in different water body

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scales. In order to address these challenges, scientists and engineers in diverse fields

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from nanotechnology, materials science, biology, hydrology to environmental

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sciences are highly suggested to communicate well and work collaboratively.

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585

Acknowledgements

586

This work was supported by the National Natural Science Foundation of China

587

(51609262, 21622404, 21621004), Chinese Postdoctoral Science Foundation

588

(2017M610161), the National Key Research and Development Program of China

589

(2016YFC0403101), and the National Science and Technology Project of China

590

(2015BAD20B03). We thank Dr. Chi Yao and Dr. Feng Li for kind proofreading.

20


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Page 21 of 27


591

592

Figures and tables

593 Pollution sources identification ATCGA…GCTAA DNA tracer1

GCTAT…AAGCC DNA tracer2

qPCR

Community

Hospital

DNA extraction Stream

Sample collection

Farm

Factory ACAGT…GTCAG DNA tracer3

594 595

CTACG…GGACT DNA tracer4

Figure 1. General concept of DNA tracer system.

596

21



Page 22 of 27

597

Non-specific

Sequence design

Random design Specific design

Specificity analysis

BLAST

Specific Chemosynthesis Biosynthesis

Synthesis

Water evaluation

Clean

Non protection

Complex

Protection

Clay Polymer Silica

Application

598 599

Figure 2. The flowchart for the preparation of DNA tracer.

22


Page 23 of 27


600 a

Encapsulation

+ DNA tracer b

Clay, polymer or silica c

Protected DNA tracer d

e

601 602

Figure 3. Protection for DNA tracer. (a) DNA tracer was protected by clay39，

603

polymer44,63 or silica65,92; (b) Attachment efficiency of DNA/RNA on clay hydrogel74;

604

(c) Protection of DNA in clay hydrogel environment against DNase. Lane 1: DNA;

605

Lane 2 and 3: DNA plus 0.01 U and 0.05 U DNase, respectively; Lane 4: clay; Lane 5:

606

DNA plus clay; Lane 6 and 7: DNA and clay plus 0.01 U and 0.05 U DNase,

607

respectively74; (d) Representative SEM image of microspheres63; (e) Representative

608

TEM image of a microsphere63.

609 610

23



Collection

Preteatment

Readout

Centrifuge

NP

qPCR

Samples P

Non-protection (Naked DNA)

Protection (Clay, polymer or silica-DNA)

Elution

Centrifuge

• 1st step: Centrifuge for 0.5 h at 13000rpm • 2nd step: Extract supernatant

• 1st step: Release DNA from clay, polymer or silica •

2nd

step: Centrifuge for 0.5 h at 13000rpm

• 3rd step: Extract supernatant

611

Page 24 of 27

Protection method DNA extraction MgCl2 and DNA Add ethylene- diaminetetraacetic were added to clay acid (EDTA) and CaCl2 Double emulsion Add chloroform or methylene method to PLA chloride Reversible DNA Add buffered oxide etch (BOE) encapsulation to silica

• 1st step: Denaturation (3min at 95 oC) • 2nd step: Denaturation (15s at 95 oC) and annealing/extension(35 s at 55 oC) • 3rd step: Fluorescence values measurement(520540nm) at the end of each cycle qPCR condition No. 0.8% agarose （1） gel(PCR) SYBR Green （2） Supermix(qPCR) SYBR Green （3） Supermix(qPCR)

612

Figure 4. A typical protocol of qPCR for the measurement of DNA tracer. (1)

613

Clay 39; (2)PLA44,63; (3)Silica92,93. NP: Non-protection; P: Protection.

24


Page 25 of 27


614

Table 1 Comparisons of DNA tracers with traditional tracers Items

Tracers

Comparisons

Detailed information

Refs

Amount in one

DT

**

The quantities of the DNA tracer used were 6~8-orders of magnitude less than the

68

experiment

TT

Tracer numbers

**

Unlimited numbers in theoretically for DNA tracer and limited numbers for traditional

TT *

The naked DNA tracer may be adsorption or degradation to natural media and

TT

*

complex environment and traditional tracers may be adsorption to natural media

Multi-source

DT

**

More effective multi-source tracing for DNA tracers due to its unlimited numbers and

tracing

TT *

TT

*

DT

*

TT

*

Detection

DT

**

sensitivity

TT

Environmental

DT

affects

TT

Rate of recovery

64,67

39,41

high sensitivity detection method compared to traditional tracers

DT

Tracing scales

38,41,42

tracers

DT

Stability

615

DT

quantity of dye, Br and chloride tracers

Both available for scales in column, stream and groundwater

61,63

1-95% for DNA tracer and 66.7-99 % for traditional tracers

44,65,91,92

Extremely sensitive for DNA tracer by using qPCR and the sensitivity for traditional

39,62

tracers are limited **

No environmental affects for DNA tracer and possible environmental pollution for

30,31,43,94,9

some traditional tracers (dyes, dissolved organic matter etc.)

5

Note: DT, DNA tracer; TT, Traditional tracer; *, equal; **, better.

25



Page 26 of 27

616 617

618

Table 2 Case studies of DNA tracers applied in water environment. Source

Type

Base

Protection

Scale

Application

Signal readout

Recovery

Refs

B S

dsDNA ssDNA

500 72

Clay /

Laboratory Groundwater

Tracing particle transport Monitoring of the multi-level samplers

PCR PCR

NM NM

39

S

ssDNA

90

/

Column and groundwater

Reproduce the flow situation of a drinking water well

qPCR

NM

62

S S S

ssDNA ssDNA ssDNA

72 80 200

/ / PLA

NM 19-55% NM

42

ssDNA

72

/

qPCR

NM

43

S

ssDNA

80

/

Batch and brook

qPCR

2.9-52.6%

64

S

ssDNA

80

Silica

Columns

qPCR

70%

65

S

ssDNA

80

/

Bedrock river

qPCR

87%

91

S

dsDNA

105

Silica

Batch

Investigate the applicability of DNA tracers Test multiple DNA tracers in hydrology Test a system for concentrating the tracers Detect connection to the sinkhole and gain an idea of the tracer arrival time Observe the behavior of DNA tracer in stream Test transport properties of SiDNAMag Test the feasibility of DNA tracing technique in a karst environment Trace the fate of silica particles in wastewater

PCR qPCR qPCR

S

Groundwater Brook Column, plot and stream Batch, column and groundwater

qPCR

95%

92

S

ssDNA

PLA

Laboratory and glacier

Test transport properties of DNA tracers

qPCR

1-66%

44

S

ssDNA

82-1 02 72

/

NM

66

dsDNA

302

Study the flow paths present in the aquifers Tracking effluent discharges in soils and groundwater

qPCR

S

River and Brook Column, lysimeter and groundwater

qPCR

NM

68

/

Note: B, Bacteriophage; S, Synthetic; NM, Not mentioned. 26


41

67 63

Page 27 of 27


619 620

TOC Graphic (300dpi, 3.19in by 1.87in)

621 622

DNA tracer has been developed for the identification of stream pollution sources. DNA tracer possesses remarkable advantages including sufficient species, specificity, environmental friendly, stable migration and high sensitivity as well as allowing for multi-points tracing.

623

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A DNA Tracer System for Hydrological Environment Investigations

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