The author(s) shown below used Federal funding provided by the U.S. 

Department of Justice to prepare the following resource: 

Document Title: Bait and Capture: Holding on to Molecules 

of Interest 

Author(s): Brian M. Kemp, Misa Winters, Cara 

Monroe, Jodi Lynn Barta 

Document Number: 251646 

Date Received: April 2018 

Award Number: 2011-DN-BX-K549 

This resource has not been published by the U.S. Department of 

Justice. This resource is being made publically available through the 

Office of Justice Programs’ National Criminal Justice Reference 

Service. 

Opinions or points of view expressed are those of the author(s) and 

do not necessarily reflect the official position or policies of the U.S. 

Department of Justice. 
Final Technical Report 

Bait and Capture: Holding on to Molecules of Interest 

Award Number: 2011-DN-BX-K549 

Prepared by Brian M. Kemp, with contributions from Misa Winters, 

Cara Monroe, and Jodi Lynn Barta 

Original submitted to the NIJ January 2015, revised July 2015.

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

1
ABSTRACT

In this study, we sought to document the efficiency of three DNA bait capture methods 

by two measures; first, in their ability to retain targeted DNA molecules and secondly in 

their ability to remove non-target DNA molecules. Efficiencies were estimated by 

comparing number of “copies in” to “copies out” with quantitative polymerase chain 

reaction (qPCR). 

The first method, “fishing” for DNA, retains only 9.06-3.53% (i.e., loss of 90.94-96.47%) 

of DNA targets ranging 109-288 base pairs (bps) in length. Minor improvement was 

achieved by employing a modified fishing protocol (i.e., shortened hybridization time, use 

of twice the amount of M-270 streptavidin-coated beads, and modified bead washing), 

resulting in average retention of 31.41-12.08% of the same set of targeted molecules. 

However, of equal concern was the inability of the method in removing more non-target 

DNA molecules than targeted molecules. The second method, primer extension capture 

(PEC), retains 15.88-2.14% (i.e., loss of 84.12-97.86%) of the same target molecules. 

Experimental modifications of PEC aimed to potentially increase this efficiency were 

generally of no avail. However, the benefit of PEC, as originally designed, is in its ability 

to remove most non-target DNA molecules (99.99%). The third method, “mega-probe” 

capture, increased the count of target molecules from the same DNA standards by 

702.46%, an impossible outcome. From our observations of negative controls, bait 

molecules became counted as captured copies when, in fact, there were no copies to 

capture. Due to unexpected experimental outcomes, we were not able to estimate the 

efficiency of this method in its removal of non-target molecules. Nevertheless, our 

principal concern about mega-probe capture is the possibility that the mega-probe bait 

becomes counted as captured target molecules. Thus, any attempt to measure its 

efficiency in retaining targeted molecules will be biased. 

Our unique approach to quantifying efficiency allowed us to begin assessing at which 

step(s) DNA is lost during the protocols tested here. We observed that most (61.35-

69.49%) of the molecules are “lost” during the essential hybridization step of the fishing 

protocol. Following the PEC protocol results in the loss of many (56.69%) of the target 

molecules prior to their immobilization on the streptavidin-coated beads (i.e., following 

extension by the polymerase and subsequent purification of the extended products). 

Experiments conducted on DNA retention following hybridization steps of the megaprobe protocol reveal tremendous loss of molecules (up to 83.04%). The losses we 

documented are attributable to: 1) the inefficiency of retaining molecules by purification 

using the Qiagen MinElute® PCR Purification Kit, 2) heat degradation of the DNA 

molecules, making them unavailable for PCR amplification, or 3) both. 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

2
TABLE OF CONTENTS

Executive Summary.......................................................... 4 

Chapter 1: How Much DNA is Lost? Measuring DNA Loss of 

STR Length Fragments Targeted by the PowerPlex 16® System 

using the Qiagen MinElute® Purification Kit........................... 11 

Chapter 2: Are We Fishing or Catching? Evaluating the 

Efficiency of Bait Capture of CODIS Fragments...................... 36 

Chapter 3: Evaluating the Efficiency of Primer Extension 

Capture as a Method to Enrich DNA Extractions....................... 58 

Chapter 4: Evaluating the Efficiency of “Mega-Probe” Capture 

as a Method to Enrich DNA Extractions................................. 79 

Dissemination of Research Findings....................................... 95 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

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3
EXECUTIVE SUMMARY 

The analysis of DNA extracted and purified from low copy number (LCN), ancient, and/or 

degraded source materials is largely complicated by: 1) the presence of contaminating 

“modern” (Kemp and Smith, 2005; Barta et al., 2014b) and “ancient” (Noonan et al.,

2005; Poinar et al., 2006) DNA, 2) co-extracted polymerase chain reaction (PCR) 

inhibitors (Alaeddini, 2011; Kemp et al., 2014a), and 3) the degree to which template 

molecules have been damaged or chemically modified post-mortem or from the time of 

deposition of the biological material (Gilbert, 2006; Alaeddini et al., 2010). As these 

associated problems make the authentication of DNA profiles from such samples 

particularly problematic, there is continued need for the development and evaluation of 

methods that increase the yield and purity of genetic material extracted from degraded 

sources. 

High throughput sequencing (HTS) of DNA [also commonly referred to as next 

generation sequencing (NGS) or massive parallel sequencing (MPS)] holds promise for 

the continued study of LCN and degraded DNA samples. While forensic DNA 

practitioners recognize its utility (e.g., Chang et al., 2009; Butler, 2010; Berglund et al.,

2011; Parson et al., 2013; Melton, 2014; Yang et al., 2014), it has already been well 

established as a reliable approach to the study ancient DNA (aDNA).

However, with short tandem repeats (STRs) as the current gold standard for forensic 

identification, it has been unclear how soon HTS would become widely used by the 

forensic DNA community, despite its advantages (Holland et al., 2009; Butler, 2010; 

Yang et al., 2014). One obvious drawback of “standard” HTS is that it is analogous to 

screening DNA prior to the invention of PCR, in that, it is not directed at molecules of 

interest, but rather represents a “shot gun” approach. Thus, standard HTS is not well 

suited for screening specific markers of interest (i.e., with much coverage at any 

particular site) or mapping repetitive units (Metzker, 2010), such as those represented by 

the STRs in the Combined DNA Index System (CODIS) panel. However, forensic 

researchers have recently demonstrated that, following STR multiplex amplification and 

indexed library preparation of those amplicons, HTS can be leveraged to produce 

reliable STR profiles of adequate depth (Zeng et al., 2015). Here coverage (or depth) 

refers to the number of molecules screened. For example, with high throughput 

sequencing if a site has two or more times coverage, the site has been observed on two 

or more independent molecules derived from the biological material from which they 

have been purified. This is vastly different than screening markers with PCR, which 

theoretically begins from a pool of molecules intact enough to contain both priming sites. 

In this case, the copy number available [e.g., as assessed by quantitative PCR (qPCR)] 

from a sample dictates the “coverage”, which is observed downstream as an average in 

the amplicons sequenced using the Sanger method or typed by other means (e.g., 

RFLP, length variance). 

As another means to leverage the power of HTS, aDNA researchers have developed a 

variety of “DNA capture” methods that allow for pre-selection of genomic regions of 

interest, which can be later subjected to HTS (e.g., Briggs et al., 2009; Maricic et al., 

2010; Carpenter et al., 2013). This is necessary because typical aDNA libraries consist 

mostly of DNA exogenous to the sample (i.e., microorganismal DNA) (Noonan et al., 

2005; Poinar et al., 2006). DNA capture in its simplest form is known as “fishing” for 

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Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

4
DNA (Anderung et al., 2008), but is also routinely referred to as “sample enrichment” or 

“pull down”. Most DNA capture methods, while variable, follow the same general 

principles. First, the DNA sample is denatured and target molecules are hybridized to 

synthetic biotinylated DNA molecules (called “probes” or “bait”). Immobilization of this 

hybridized complex on streptavidin coated magnetic beads effectively “captures” the 

targets. A strong magnet is used to attract the beads to the side or bottom of the tube. 

Buffers are used to wash away impurities and non-target DNA (i.e., genomic regions not 

of interest from the organism under investigation, as well as any exogenous DNA), and 

then the beads are released from the magnet and the target DNA disassociated from the 

probes. In the end, there should be enrichment of target over non-target DNA 

molecules, and it should also be free of impurities (e.g., PCR inhibitors). 

While most DNA capture methods have been developed to enrich from aDNA libraries 

(i.e., already amplified DNA) that typically contain comparatively little endogenous DNA 

(Carpenter et al., 2013), it is possible that similar methods could be used to capture 

target molecules from a sample’s total DNA eluate, to prescreen and enrich for DNA 

markers of interest prior to PCR amplification or even library builds. 

Taking this perspective, it is essential to further consider that when targeting CODIS 

markers (or any set of markers) by traditional PCR, that these reactions contain far more 

non-target genomic DNA of the individual than target DNA [not to mention the possible 

complicating factor of non-target exogenous DNA associated with the sample (Noonan 

et al., 2005; Poinar et al., 2006)]. For example, the sum of the average amplicon lengths 

targeted in Promega Male 9948 DNA by the Promega PowerPlex 16® System is 3573 

base pairs (bps) (Promega, 2008; Kemp et al., 2014b) (see Chapter 1). Given that the 

human genome is ~3.2 billion bps in length, only ~1.12x10-4% of it need be present in a 

DNA extract to generate a complete profile. In other words, the vast majority of the 

genome is of no consequence to generating a full CODIS profile. In general, 

amplification efficiency of targeted DNA is sub-optimal because non-target DNA can 

interfere between polymerase and targeted DNA molecules and/or compete as binding 

sites for primers (Wilson, 1997; Nielsen et al., 2008). If “capture extracted” DNA is 

efficient, it could possibly lead to more reliable DNA profiling. 

The goal of our research was to document, by two measures, the efficiency of three 

DNA capture methods [“fishing for DNA” (following Anderung et al., 2008), “primer 

extension capture (or PEC)” (following Briggs et al., 2009), and a method we have come 

to call “mega-probe capture” (following Maricic et al., 2010)] in their abilities to, first,

retain targeted DNA molecules (fragments of DNA that contain the Promega PowerPlex 

16® System markers) and, secondly, in their ability to remove non-target DNA molecules 

[Northern fur seal (Callorhinus ursinus) mitochondrial DNA (mtDNA) fragments] from a

pool containing both. We specifically chose northern fur seal mtDNA as non-target 

because in our prior experience it served well as a system that could be reliable 

quantified with quantitative PCR (qPCR) (Winters et al., 2011; Barta et al., 2013; Barta et 

al., 2014a; Barta et al., 2014b). 

To achieve this goal, efficiencies were estimated following a qPCR approach of 

comparing numbers of “copies in” of DNA standards to those that are retained by the 

particular method, or “copies out” (Barta et al., 2014b; Kemp et al., 2014b) (see also 

Chapter 1). Our approach to this matter is rather unique, in that it allows each method to 

be evaluated relative to 100% efficiency and in direct relation to one another. 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

5
 

The results of these efforts are summarized below and detailed in the four chapters that 

follow this executive summary.

Chapter 1: How Much DNA is Lost? Measuring DNA Loss of STR Length 

Fragments Targeted by the PowerPlex 16® System using the Qiagen MinElute 

Purification Kit 

In this chapter we established our methodology of using qPCR to estimate “copies in” 

and “copies out”, which was a critical step for conducting the experiments reported in the 

remaining chapters. Moreover, we took advantage of his period of the grant to estimate 

the degree of DNA loss associated with purification with the Qiagen MinElute® PCR 

Purification Kit, which was found to be particularly useful in later experiments to account 

for such loss of DNA. In addition, we designed these experiments to fill in some of the 

gaps in knowledge that remained from experiments conducted during our last NIJ grant 

["NIJ Proposal to Enhance Methods for Studying Degraded DNA" 2008-DN-BX-K008]

(Barta et al., 2014b). 

The success in recovering genetic profiles from aged and degraded biological samples

is diminished by fundamental aspects of DNA extraction, as well as its long-term

preservation, that are not well understood. While numerous studies have been 

conducted to determine whether one extraction method performed superior to others, 

nearly all of them were initiated with no knowledge of the actual starting DNA quantity in 

the samples prior to extraction, so they ultimately compared the outcome of all methods 

relative to the best (Barta et al., 2014b). Using qPCR to estimate the copy count of 

synthetic standards before (i.e., “copies in”) and after (i.e., “copies out”) purification 

(Barta et al., 2014b) by the Qiagen MinElute® PCR Purification Kit, we documented 

DNA loss within a pool of 16 different sized fragments ranging from 106-409 base pairs

(bps) in length, corresponding to those targeted by the Promega PowerPlex 16® System.

Across all standards starting from 104 to 107 copies/L, loss averaged 21.75% to 60.56% 

(mean 39.03%), which is not congruent with Qiagen’s (2008) claim that 80% of DNA 

fragments 70 bp to 4 kb in length are retained using this product (i.e., a 20% loss). Our 

study also found no clear relationship between either DNA strand length and retention or 

starting copy number and retention. This suggests that there is no molecule bias across 

the MinElute® column membrane and highlights the need for manufacturers to clearly 

and accurately describe how their claims are made, and should also encourage 

researchers to document DNA retention efficiencies of their own methods and protocols.

As reported in this chapter, understanding how and where to reduce loss of molecules

during extraction and purification will serve to generate clearer and more accurate data, 

which will enhance the utility of ancient and low copy number DNA as a tool for closing 

forensic cases or in reconstructing the evolutionary history of humans and other 

organisms. 

Chapter 2: Are We Fishing or Catching? Evaluating the Efficiency of Bait Capture 

of CODIS Fragments 

The experiments reported in this chapter were conducted to document the efficiency of 

fishing for DNA, a method described by Anderung et al (2008), one that forms the basis 

for the other two capture methods evaluated in this project.

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

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necessarily reflect the official position or policies of the U.S. Department of Justice.

6
Following the rationale of the fishing method, we converted Promega PowerPlex 16® 

System primers into biotinylated probes, and subsequently used to them capture target 

DNA molecules (see Figure 1 in Chapter 2). Retention of DNA targets ranging 109-288 

base pairs (bps) in length was only 9.06-3.53% (i.e., loss of 90.94-96.47%) using the

fishing protocol as previously described. Minor improvement was achieved by 

employing a modified fishing protocol (i.e., one with a shortened hybridization time, use 

of twice the amount of M-270 streptavidin-coated beads, and modified bead washing), 

resulting in average retention of 31.41-12.08% of the same set of targeted molecules. 

Of equal concern was the inability of the fishing method in removing more non-target 

DNA molecules than targeted molecules. In other words, fishing for DNA simply lowered 

the concentration of DNA in the extract. 

We observed that most (61.35-69.49%) of the molecules are “lost” during the essential 

hybridization step of the fishing protocol. A possible explanation for the loss during

hybridization is that heat treatment degrades the DNA molecules, making them nonamplifiable, as they no longer contain both priming regions, a requisite for PCR 

amplification. This rather novel observation will require follow-up studies, ones that 

could form the basis for a future project funded by the NIJ. 

While the fishing method may be useful in the study of DNA compromised by 

polymerase chain reaction (PCR) inhibitors (Wang and McCord, 2011), the results 

presented in this chapter will allow others to carefully weigh this possible advantage 

against the degree of expected DNA loss and the non-selectivity of the method for 

targeted over non-targeted DNA.

Chapter 3: Evaluating the Efficiency of Primer Extension Capture as a Method to 

Enrich DNA Extractions 

The experiments reported in this chapter were conducted to document the efficiency of 

primer extension capture (PEC), a method described by Briggs et al. (2009). The PEC 

method expands on fishing for DNA with the addition of one cycle of annealing and 

polymerase extension of the biotinylated probes on the target molecules (see Figure 1 in 

Chapter 3). Following extension, the biotinylated probes become complementary to the 

length of the target molecule. Thus, the target DNA should be more tightly bound to the 

probes, in comparison to fishing for DNA with short probes. 

Conducted as previously described, PEC retention of DNA targets ranging 109-288 base 

pairs (bps) in length was only 15.88-2.14% (i.e., loss of 84.12-97.86%). Experimental 

modifications of PEC aimed to potentially increase this efficiency were generally of no 

avail. However, the benefit of PEC, as originally designed, is in its ability to remove 

most non-target DNA molecules (99.99%). This is precisely why it can be of value in 

focusing the power of HTS on molecules of interest over non-target regions of the 

genome and also act to exclude DNA exogenous to the individual under study. 

Regardless of absolute efficiency, PEC is probably more efficient at removing non-target 

DNA than fishing because, even if the probes were able to anneal to non-target 

molecules, mismatches at or near the 3’ ends of the primers would result in poor 

extension of the molecule by the polymerase (Palumbi, 1996). Thus, they would be 

subsequently more difficult to immobilize.

In this set of experiments we also documented the loss of many (56.69%) of the target 

molecules prior to their immobilization on the streptavidin-coated beads, that is, following 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

7
extension and purification of the extended products. This loss can be attributed first to 

degradation of the molecules caused by heat treatment (i.e., causing them to not be 

countable during qPCR), which is exacerbated by the loss attributed to purification by

the Qiagen MinElute® PCR Purification Kit. 

The results presented in this chapter will allow researchers to carefully weigh the general 

inefficiency of PEC in retaining target molecules over its high efficiency in removing nontarget molecules. Moreover, these efficiencies are directly comparable to those 

observed following fishing for DNA (Chapter 2), or any alternative method for DNA 

enrichment. 

Chapter 4: Evaluating the Efficiency of “Mega-Probe” Capture as a Method to 

Enrich DNA Extractions 

The experiments reported in this chapter were conducted to document the efficiency of a 

DNA capture method described by Maricic et al (2010). Their method converts long 

DNA fragments into biotinylated probes that are subsequently used to capture other 

DNA molecules, those complementary to the long probes (see Figure 1 in Chapter 4). 

We refer to this approach as “mega-probe” capture because it employs biotinylated 

probes much longer than those used in both fishing and PEC. 

Mega-probe capture increased target molecules from standards by 702.46%, an 

impossible outcome. From our observations of negative controls, 6217.43, 14555.55,

and 36621.00 copies/μL of D16s539, D13s317, and amelogenin bait molecules, 

respectively, became counted as captured copies when, in fact, there were no copies to 

capture. Due to unexpected experimental outcomes, we were not able to estimate the 

efficiency of this method in its removal of non-target molecules. Nevertheless, our 

principal concern about mega-probe capture is the possibility that the mega-probe bait 

becomes counted as captured target molecules. Thus, any attempt to measure its 

efficiency in retaining targeted molecules will be biased. 

Additional experiments conducted on DNA retention following hybridization steps of the 

mega-probe protocol reveal tremendous loss of molecules (up to 83.04%) attributable to 

both purification by the Qiagen MinElute® PCR Purification Kit and heat exposure. 

These experiments highlight the need for those working with LCN and degraded DNA 

samples to be mindful of the loss of already limited DNA strands available for analysis 

due to the experimental treatments used to study them. 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

8
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Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

10
 

Chapter 1: How Much DNA is Lost? Measuring DNA Loss of

STR Length Fragments Targeted by the PowerPlex 16® System 

using the Qiagen MinElute® Purification Kit 

[Note: A similar version of this paper has been published in volume 86(4) of the 

journal Human Biology. Revisions made to this final technical report occurred

after the publication date.] 

Brian M. Kempa,b, Misa Wintersa,b, Cara Monroea, and Jodi Lynn Bartac

a Department of Anthropology, Washington State University, Pullman, WA 99164-4236 

b School of Biological Sciences, Washington State University, Pullman, WA 99164-4910 c Department of Biological and Health Sciences, Madonna University, Livonia, MI 48150

Corresponding Author: 

Brian M. Kemp 

Department of Anthropology 

Washington State University 

Pullman, WA 99164 

Office: 509-335-7403 

Fax: 509-335-3999 

bmkemp@wsu.edu

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

11
ABSTRACT

The success in recovering genetic profiles from aged and degraded biological samples

is diminished by fundamental aspects of DNA extraction, as well as its long-term

preservation, that are not well understood. While numerous studies have been 

conducted to determine whether one extraction method performed superior to others, 

nearly all of them were initiated with no knowledge of the actual starting DNA quantity in 

the samples prior to extraction, so they ultimately compared the outcome of all methods 

relative to the best. Using quantitative PCR (qPCR) to estimate the copy count of 

synthetic standards before (i.e., “copies in”) and after (i.e., “copies out”) purification by 

the Qiagen MinElute PCR Purification Kit, we documented DNA loss within a pool of 16 

different sized fragments ranging from 106-409 base pairs (bps) in length, corresponding 

to those targeted by the Promega PowerPlex 16® System. Across all standards starting 

from 104 to 107 copies/L, loss averaged between 21.75% and 60.56% (mean 39.03%), 

which is not congruent with Qiagen’s claim of 80% retention of DNA fragments 70 bp to 4 

kb in length (i.e., 20% loss). Our study also found no clear relationship between either 

DNA strand length and retention or starting copy number and retention. This suggests 

that there is no molecule bias across the MinElute column membrane and highlights the 

need for manufacturers to clearly and accurately describe how their claims are made, 

and should also encourage researchers to document DNA retention efficiencies of their 

own methods and protocols. Understanding how and where to reduce loss of molecules 

during extraction and purification will serve to generate clearer and more accurate data, 

which will enhance the utility of ancient and low copy number DNA as a tool for closing 

forensic cases or in reconstructing the evolutionary history of humans and other 

organisms. 

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12
It has been a mere 25 years since the first demonstrations that bones can contain 

preserved DNA even many hundreds to thousands of years following death of the 

organism (Hagelberg et al., 1989; Horai et al., 1989; Montiel et al., 2007). Shortly 

afterwards, forensic DNA researchers demonstrated the usefulness of obtaining genetic 

profiles from skeletal remains (Hagelberg and Clegg, 1991; Hagelberg et al., 1991; 

Hochmeister et al., 1991; Stoneking et al., 1991; Jeffreys et al., 1992) and today this 

type of analysis is indispensable to the field (Edson et al., 2004; Milos et al., 2007; 

Edson et al., 2009; Mundorff et al., 2009; Caputo et al., 2013; Ambers et al., 2014a; Blau 

et al., 2014; Mameli et al., 2014). Amazingly, DNA has now been recovered from bones 

dating from 300,000 to 780,000 years old (Dabney et al., 2013; Meyer et al., 2013; 

Orlando et al., 2013) and complete genomes are being routinely sequenced from ancient 

specimens (Green et al., 2010; Rasmussen et al., 2010; Reich et al., 2010; Raghavan et 

al., 2014; Rasmussen et al., 2014; Rasmussen et al., 2015).

However, the success in recovering genetic profiles from aged and degraded biological 

samples, including bones, needs to be balanced against the sobering reality that there 

are still fundamental aspects of long-term DNA preservation that are not well 

understood, aspects that need to be further approached through experiments with 

simple and clear methodologies. To illustrate this point, one recent study posed the 

question “DNA in ancient bone – Where is it located and how should we extract it?” 

(Campos et al., 2012). This seemingly straightforward question had no easy answer, 

rather, it raised a high degree of speculation over whether the organic or inorganic 

portions of the bone are superior in their preservation of DNA. As is often the case, this 

study raised more issues about the mechanics of DNA preservation than it resolved. In 

addition, despite years of being convinced that DNA preservation is positively correlated 

with bone density (Parsons and Weedn, 1997; NIJ, 2005; Prinz et al., 2007), two recent 

studies clearly bring that relationship into question (Mundorff and Davoren, 2013; Barta 

et al., 2014a). Even more fundamentally, there is no way to know how much DNA exists 

in bone samples, given that one can only observe how much is retained following the 

extraction and purification processes that are known to induce loss (e.g., Barta et al., 

2014b). This parallels the questioning by van Oorschot and colleagues (2003) of how 

much DNA is actually available from touched objects, when resulting extracts do not 

contain all of the DNA molecules that were originally present on the objects. In fact, this 

reasoning has led to vast improvements in DNA collection swabs (Marshall et al., 2014). 

DNA recovered from aged and degraded biological samples has long been observed to 

be in low copy number (LCN), and is degraded to short strand lengths (Pääbo et al., 

1988; Hagelberg et al., 1989; Pääbo, 1989). This is likewise true for many trace DNA or 

touch DNA samples (Lowe et al., 2002; Hudlow et al., 2010; van Oorschot et al., 2010). 

As such, it is hardly surprising that numerous efforts have been directed toward 

determining the best method of DNA extraction (i.e., to retain the most amount of DNA). 

Many studies have demonstrated that the performance of one extraction method was 

superior to others tested for some tissue type(s), ranging in age and state of 

preservation (Cattaneo et al., 1997; Yang et al., 1998; Hoff-Olsen et al., 1999; Castella 

et al., 2006; Davoren et al., 2007; Loreille et al., 2007; Rohland and Hofreiter, 2007b;

Rohland et al., 2010), or compared retention of DNA from the organic portion of bone

with that from the inorganic portion (Schwarz et al., 2009; Campos et al., 2012). Yet, 

these studies began with no knowledge of the actual DNA quantity in the samples prior 

to extraction, so they ultimately compared the outcome of all methods relative to the 

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13
best. While this approach can result in the identification of a best method, it cannot 

determine how well the method performs against 100% recovery.

Manipulation of samples containing DNA will result in DNA loss. This is true regardless 

of whether this loss is due to not swabbing all DNA present on a touched object in the 

first place, or losing DNA in any of the many subsequent steps used during extraction 

and purification. Recently, researchers have begun to address these issues by 

attempting to measure the degree of DNA loss, relative to a standard, associated with 

used of various DNA collection swabs (Marshall et al., 2014) and DNA extraction 

methods (Lee et al., 2010; Dabney et al., 2013; Barta et al., 2014b) 

Lee and colleagues (2010) artificially degraded human genomic DNA with DNAse I and 

diluted this to 25 ng standards. They also created non-degraded standards at the same 

concentration. Standard concentrations were determined via quantitative PCR (qPCR) 

using the Quantifiler® Human DNA Quantification Kit (Life Technologies, Carlsbad, CA, 

USA). These standards were then subjected to three extraction methods utilizing 

Qiagen (Venlo, Limburg, Netherlands) products: 1) QIAamp Mini Kit, which employs 

QIAamp Mini Spin Columns, 2) QIAquick PCR purification kit , which employs QIAquick 

Spin Columns, and 3) QIAamp Mini spin columns combined with reagents from the 

QIAquick PCR purification kit (Buffers PB and PE). While they found that, on average, 

the third method performed best (retaining 50.8% and 38.9% of the degraded and intact 

standards, respectively), there was little difference in comparison to the other two 

methods. Surprisingly, on average 0.7%-11.9% more of the degraded standards were 

retained in comparison to the intact ones. While it was not possible to assess 

degradation in strand length caused by Lee and colleagues’ (2010) experimental 

modification with DNAse I treatment (i.e., the authors did not run the degraded samples 

on a gel to observe the resulting strand lengths), 25 ng of genomic DNA equates to over 

7200 copies of each of the ~3.2 billion nucleotides that the nuclear genome comprises 

(ignoring the collective nucleotide count per cell that the mitochondrial genomes 

contain). This is equivalent to the amount of nuclear DNA found in 3600 diploid cells. 

Regarding copy number, mitochondrial DNA (mtDNA) retrieved from most ancient 

samples has typically crossed several orders of magnitude from 10 to 102 copies/μL 

(Malmstrom et al., 2005; Poinar et al., 2006; Malmstrom et al., 2007; Schwarz et al., 

2009; Winters et al., 2011) with a few mammoths and dogs at 103 copies/μL (Malmstrom 

et al., 2007; Schwarz et al., 2009) and one unusual dog sample that yielded 105

copies/μL (Malmstrom et al., 2005). As expected, ancient nuclear DNA has been 

observed at hundreds to thousands of times fewer copies/μL compared to mtDNA 

(Schwarz et al., 2009). While forensic researchers may not require a definition for LCN 

(Gill and Buckleton, 2010) and the threshold may depend, in part, on the technology 

used to type sample (Budowle et al., 2009), one such description by the National 

Forensic Science Technology Center (Largo, FL, USA) DNA Analyst Training manual 

refers to LCN as “the examination of less than 100 picograms of input DNA”, further 

stating that “assuming 3.5 pg of DNA per haploid cell, [this quantity] is equivalent to 

approximately 15 diploid or 30 haploid cells” (http://www.nfstc.org). Budowle and 

colleagues (2009) review evidence that less than 200 pg of input DNA should be 

considered LCN, importantly recognizing that it is really the stochastic threshold of which 

one should be mindful. 

To estimate the degree of DNA loss, Dabney and colleagues (2013) subjected a 

standard mixture of five NoLimit DNA fragments (ThermoScientific, Waltham, MA, USA) 

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14
[35, 50, 75, 100, and 150 base pairs (bps)] at a concentration of 5.7 ng/μL to the 

extraction method of Rohland and Hofreiter (2007a) and a modified version of that 

protocol. The modifications included a change to binding buffer composition, buffer 

volume, and replacement of loose silica for a fixed silica column (Qiagen MinElute spin 

column). DNA loss was quantified against the standard using a BioAnalyzer with a 1000 

DNA chip (Agilent, Santa Clara, CA, USA). This represents a particularly creative 

approach, as this method can simultaneously estimate DNA loss across various sized

fragments. However, the 1000 DNA chip has a low-end detection of ≥0.5 ng/μL. 

Evaluated against their standard concoction, this equates to ~5.6 billion total copies/μL 

(or ~1.12 billion copies/μL of each sized fragment) (see discussion by Barta et al., 

2014b). This would make it necessary to evaluate loss of very high copy number 

standards, as they chose to do so, starting at ~64 billion total copies/μL (or ~12.8 billion 

copies/μL of each sized fragment). In this case, a loss of ≥99.9999% of the standards

employed by Dabney and colleagues (2013) would be required to result in ≤104

copies/μL, a range typically observed in aDNA studies and those of LCN forensic 

samples, making it difficult to assess the applicability of their results in these instances. 

Nevertheless, Dabney and colleagues (2013) observed that the Rohland and Hofreiter 

(2007a) method was associated with 72% and 22% retention of 150 bp and 35 bp 

fragments, respectively. Their modified extraction protocol resulted in the opposite 

relationship, with ~84% and 95% retention of these fragments, respectively [estimated 

from Figure 1 of Dabney and colleagues (2013)].

Lastly, Barta and colleagues (2014b) used qPCR to estimate DNA loss of a single sized 

DNA fragment (181 bps) at concentrations of 102 to 104 copies/μL (~130-50000 

copies/μL) associated with common extraction methods, including phenol:chloroform, 

alcohol precipitation, microconcentration, and silica-based extractions. They determined 

that methods which employ numerous steps, for example that of Kemp and colleagues 

(2007), compound DNA loss, which can result in less than 0.5% retention of the 

molecules. Simple silica based methods [Wizard® PCR Preps Purification System 

(Promega, Madison, WI, USA) and QIAquick PCR purification kit] were associated with 

~36-39% retention of the 181 bp standard. One drawback of the Barta and colleagues’ 

(2014b) study was that their standard contained DNA fragments of only a single size.

Clearly, the differences among the methodological approaches and results obtained in 

the three studies just described underscores the need for further research directed at 

understanding DNA retention efficiencies. Thus, the object of the current study is to 

document DNA loss within a pool of 16 different sized fragments, corresponding to those 

targeted by the Promega PowerPlex 16® System. Ultimately, we followed the procedure 

of Barta and colleagues (2014b) in using qPCR to estimate DNA loss by comparing 

standards before (i.e., “copies in”) and after (i.e., “copies out”) purification, in this case 

with the Qiagen MinElute PCR Purification Kit. This permitted us to evaluate the 

relationship between DNA strand length and retention, and also that of starting copy 

number and retention.

MATERIALS AND METHODS

System Choices 

The Promega PowerPlex 16® System was chosen to create standards because it targets 

the thirteen Combined DNA Index System (CODIS) markers, in addition to the 

amelogenin, Penta D, and Penta E markers. The amplicons produced from the 

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15
 

Promega 9948 Male DNA sample range in size from 106 bps of the Amelogenin gene on 

the X chromosome to 428 bps from the Penta D locus on chromosome 15 (Table 1). 

Important to this experimental design is that the PowerPlex 16® System is validated for 

casework and the primer sequences are published (Table 1) (Masibay et al., 2000; 

Krenke et al., 2002; Butler et al., 2003), which was essential to constructing the 

standards, as described below. 

Qiagen’s (2008: pg 8) claim that MinElute columns retain 80% of fragments ranging from 

70 bp to 4 kilobases (kb), and that the membrane was made purposely for elution in 

volumes as small as 10 μL, makes the MinElute PCR Purification Kit a common choice 

for use in library purification and/or enrichment protocols for high throughput sequencing 

(HTS) (e.g., Briggs et al., 2009; Maricic et al., 2010; Carpenter et al., 2013; Enk et al., 

2013; Warinner et al., 2014). It is being increasingly used in the purification of DNA from 

ancient samples (Ginolhac et al., 2012; Carpenter et al., 2013; Dabney et al., 2013; Meiri 

et al., 2013), suboptimal samples [e.g., bones removed from aged owl pellets (Buś et al., 

2014)], and also in forensic studies (Coble et al., 2009; Loreille et al., 2010; Ambers et 

al., 2014a). The columns have also been employed by forensic DNA researchers in 

modified extraction methods (Marshall et al., 2014). 

The components of the Qiagen MinElute PCR Purification Kit reveal that it is based on 

binding DNA to a fixed silica column [versus using loose silica, such as in the Promega 

Wizard® PCR Preps Purification System or the Rohland and Hofreiter (2007a) method], 

from which DNA is eluted after first washing with alcohol. Based on the Material Safety 

Data Sheets (MSDS), Buffer PB is a mixture of 25-50% guanidinium chloride (or 

hydrochloride, GuHCl) and 25-50% isopropanol. According to Qiagen, “Buffer PB 

contains a high concentration of guanidine hydrochloride and isopropanol. The exact 

composition of Buffer PB is confidential” and “The composition of Buffer PE is 

confidential” (www.qiagen.com). OpenWetWare (www.openwetware.org) states that 

Buffer PB is 5 M GuHCl and 30% isopropanol and Buffer PE is 10 mM Tris-HCl pH 7.5 

and 80% ethanol. Buffer EB is 10 mM Tris-Cl, pH 8.5 (www.qiagen.com). In general, 

most silica-based extraction methods, including the Qiagen MinElute PCR Purification 

Kit, are minor variants of that described by Boom and colleagues (1990). 

For these reasons, we thought the Qiagen MinElute PCR Purification Kit represented a 

well-used product that would benefit researchers when subjected to tests for DNA loss. 

Creating Individual Standards and Calculation of qPCR Efficiency (E) for 

Individual Markers 

Each of the sixteen fragments of the genome targeted by the PowerPlex 16® System

were individually amplified eight times in 30 L PCRs containing: 0.32 mM dNTPs, 1X

PCR buffer, 1.5 mM MgCl2, 0.24 L primers (Table 1), 0.6 U Platinum Taq DNA 

Polymerase (Life Technologies), and 1.5 μL of male DNA (Promega 9948). PCR 

negatives accompanied these reactions to monitor for contamination. Cycling was 

performed with an initial 3 minute hold at 94°C followed by 40 cycles of 15 second holds 

at 94°C, 60°C, and 72°C, followed by a 3 minute hold at 72°C. Successful amplification 

was confirmed by separating 4 μL of PCR product on 2% agarose gels, visualized with 

ethidium bromide staining under UV illumination.

The remaining volumes of each set of eight reactions were pooled and purified with the 

Qiagen QIAquick PCR Purification Kit following the manufacturer’s protocol except that 

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16
 

the pH indicator was not added and the final elution was conducted with molecular grade 

water. Following purification of the amplicons, standard concentration was determined 

by taking the average of 2-3 spectrophotometry readings using a Nanodrop 

(Thermoscientific), from which copy numbers were calculated as follows:

1. The average weight of a base pair (bp) is 650 Daltons. The molecular weight of the 

amplicons from each of the 16 pools was estimated by taking the product of 650 and 

their bp length (Table 1). Where the Promega 9948 Male DNA is heterozygous at 11 of 

the PowerPlex 16® System markers, we used the mean length of the amplicon sizes.

The inverse of the molecular weight is the number of moles of template present in one

gram of material.

2. Using Avogadro's number of 6.022 ×	1023 molecules/mole, the number of molecules 

of the template per gram can be calculated as: mol/g * molecules/mol = molecules/g

3. Finally, the number of molecules in the purified pool of PCR products can be 

estimated by multiplying by 1 ×	10-9 (g/ng) to convert to ng and then multiplying by the 

concentration of the template (ng/μL). 

This calculation requires the user to input the concentration of the template present in 

ng/μL determined by spectrophotometry along with the length of the DNA molecules (in 

bps), and with this information the number of copies of the template can be calculated 

using the following: 

number of copies = [amount (ng) * 6.022 ×	1023] / [length (bp) * 1 ×	109 * 650] 

Following calculation of the number of copies of each pool of amplicons, each was 

diluted to a volume containing 1 ×	109 copies/μL. From these, a series of ten dilutions at 

1 ×	105, 1 ×	104, 5 ×	103, 2 ×	103, 1 ×	103, 5 ×	102, 2.5 ×	102, 1 ×	102, 5 ×	101, and 1 ×	101

copies/μL were created.

Quantitative PCRs were performed in an Applied Biosystems 7300 Real Time PCR 

System (Life Technologies). Twenty five μL qPCR reactions for FGA, D8S1179, vWA, 

and TPOX contained 0.256 mM dNTPs, 0.96X PCR Buffer, 3 mM MgCl2, 0.2X SYBR

Green, 0.5 mM Rox, 0.4uM of each primer, 0.5 U of Platinum Taq DNA Polymerase, 

and 5 μL of standard DNA. For the remaining twelve markers, 25 μL qPCR reactions 

contained: 12.5 μL SYBR Green Real-Time PCR Master Mix (Life Technologies), 0.5 

mM Rox, 0.4uM of each primer, and 5 μL of standard DNA. Cycling conditions were as 

follows: 50°C for 2 min, 95°C for 3 min, followed by fifty cycles of 95°C for 15 sec, 60°C 

for 30 sec, 72°C for 30 sec, and then a dissociation step of 95°C for 15 sec and 60°C for 

1 min. The efficiency (E) and coefficient of determination (R2) were determined from 

standard curves, created from four to six reactions each from the ten dilutions. Four no 

template controls (NTCs) accompanied each set of reactions to monitor for the presence 

of contamination.

Creating Pooled Standards and Calculation of qPCR Efficiency (E) for Individual 

Markers Within the Pool 

The sixteen amplicons were pooled and diluted to 1 ×	107, 1 ×	105, 1 ×	104, 5 ×	103, 2 ×	

103, 1 ×	103, 5 ×	102, 2.5 ×	102, 1 ×	102, 5 ×	101, 1 ×	101 copies/μL. For example, the 1 ×	

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17
105 dilution contained 1 ×	105 copies of each of the sixteen amplicons per μL (totaling 

1.6 ×	106 total amplicons per μL). Quantitative PCR was conducted using the SYBR

Green Real-Time PCR Master Mix as described above. The E and R2 for each of the 

sixteen reactions were determined from standard curves, created from four to six 

reactions each from ten dilutions ranging 1 ×	105 to 1 ×	101 copies/μL. Four no template 

controls (NTCs) accompanied each set of reactions to monitor for the presence of 

contamination. 

Evaluating DNA Loss Associated with the Qiagen MinElute PCR Purification Kit 

DNA loss associated with use of the Qiagen MinElute PCR Purification Kit was 

estimated from the 1 × 107, 1 × 105, and 1 × 104 pooled standards as follows: 

1. A 400 μL aliquot of Buffer PB was added to 80 μL of each pooled standard (i.e., 

copies in) and mixed by inversion. 

2. Each mixture was transferred to a MinElute column that was placed over a 2 mL 

collection tube. The tubes were centrifuged at 16,100 x g for 1 min in a fixed angle 

Eppendorf 5415D model centrifuge.

3. Flow-though was discarded and the MinElute columns were placed back into the 

collection tube. 

4. A 750 μL aliquot of Buffer PE was added to each MinElute column and the tubes 

were centrifuged for 1 minute at 16,100 x g. 

5. Flow-though was discarded and the MinElute columns were placed back into the 

collection tube. The tubes were centrifuged for 1 minute at 16,100 x g. 

6. The MinElute columns were placed in clean 1.5 mL tubes to which 80 μL of 10mM 

Tris-HCl, ph 8.5 was added to the center of the column membrane. This was left at

room temperature for 1 minute. 

7. The tubes were centrifuged for 1 minute at 16,100 x g. 

Extraction negatives were conducted in parallel with the standards to monitor the 

appearance of any contamination. The final 80 μL volumes contained molecules 

retained from the extraction process (i.e., copies out). Since the Penta D qPCR assay 

behaved stochastically and at times very poorly, both when analyzed individually, as well 

as when pooled (Table 1), this fragment was excluded from further analysis. 

Quantification of the other 15 markers retained in 1 ×	107 extracted standards was 

conducted in duplicate against standard curves, generated from two reactions each of 1 

×	107, 1 ×	105, 1 ×	104, 5 ×	103, 2 ×	103, 1 ×	103, 5 ×	102, 2.5 ×	102, and 1 ×	102. Two 

additional 1 ×	107 standards were quantified as unknowns in order to monitor the 

concentration of the pre-extracted standard. Quantification of the molecules that 

remained in the 1 ×	105 and 1 ×	104 extracted standards was similarly conducted, but 

against standard curves generated from 1 ×	105, 1 ×	104, 5 ×	103, 2 ×	103, 1 ×	103, 5 ×	

102, 2.5 ×	102, and 5 ×	101 reactions. Two additional 1 ×	105 and 1 ×	104 standards were 

also quantified as unknowns in order to monitor the concentration of these pre-extracted 

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18
standards. Quantitative PCR reactions were conducted with SYBR Green Real-Time 

PCR Master Mix as described above. 

Calculating Efficiency of DNA Retention 

Subtraction of the number of “copies out” (measured as the average of duplicate qPCR 

amplifications from the molecules retained following extraction) from “copies in” 

(measured as the average of duplicate qPCR amplifications of the standards treated as 

unknowns) divided by “copies in” multiplied by 100 provides the percent efficiency (or 

percent retention of molecules of each experimental method: [(copies in - copies out)/

copies in] *100=efficiency). One hundred minus efficiency provides a measure of 

percent loss. Loss of each of the 15 markers was determined in this manner 3-4 times, 

from which the average loss and its associated uncorrected standard deviation (i.e., the 

standard deviation of the sample) were calculated. 

Linear regression of percent DNA loss against base pair length and starting copy 

number was conducted in StatPlus. An alpha value of 0.05 was used as the cut-off for 

statistical significance.

RESULTS 

Individually screened, the qPCR efficiencies for 15 markers ranged from 75.81-104.12% 

(Table 1). The Penta D qPCRs behaved unpredictably, and at best achieved an 

efficiency of 68.05%. Screened within a pool, qPCR efficiencies for 14 markers ranged 

from 86.69-100.81%. While the efficiency of the D21S11 reaction was 64.33%, we 

proceeded to evaluate loss of these amplicons with the intention of omitting the results if 

the efficiencies did not improve. The efficiency of Penta E was 84.30%, but due to its 

large fragment size, this was not considered unacceptable, and the copy numbers 

relevant to the standards were fairly consistent. 

The efficiencies of all subsequent qPCRs used to evaluate DNA loss ranged from 82.27-

96.53% and the R2 values from 0.9918-0.9995 (Appendix A). It is notable that the three 

D21S11 qPCR reactions had efficiencies of 89.89-91.46%, suggesting that 

quantifications from these reactions are reliable.

Across the trials, average DNA loss of the 1 ×	104 standards ranged from 34.68% (SD 

5.2%) to 60.56% (SD 1.84%), the 1 ×	105 standards ranged from 34.83% (SD 5.25%) to 

54.28% (SD 4.72%), and the 1 ×	107 standards ranged from 21.75% (SD 2.7%) to 41.17 

(SD 1.86%) (Appendix A at the end of this chapter, Table 2). While the shortest DNA 

fragment (Amelogenin, average 109 bps) was associated with the greatest percentage 

loss across all of the standards, the slopes between DNA fragment size (106-409 bps) 

are no different than zero (i.e., p-values are greater than 0.05) (Figure 1). While there is 

an inverse relationship between starting copy (104, 105, 107) and average loss (see

Table 2, 42.95%, 41.72%, 32.44%), the slope is not different from zero (p=0.063). 

DISCUSSION 

All steps in the extraction and purification of DNA from biological materials will result in 

some loss of DNA. While the degree of loss associated with various manipulations is 

largely unknown, it is important to at least have an estimation, which was the goal of this 

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19
study. For example, LCN is an expectation for DNA derived from ancient samples. In

fact, this is one of numerous characteristics of aDNA that are used for convincing others 

of the authenticity of one’s results (Cooper and Poinar, 2000; Pääbo et al., 2004). 

However, the potential degree to which researchers may create the condition, then use 

that condition to authenticate their results has only recently come to light (Barta et al., 

2014b). More important than arguing what are acceptable copy numbers for aDNA 

samples or how LCN is to be defined, is advocating a wide spread recognition that large 

numbers of precious DNA copies are inadvertently discarded during the extraction and 

purification processes. This parallels closely with the message conveyed by van 

Oorschot and colleagues (2003) following their realization that not all of the DNA present 

on touched objects is recovered. Given that their observation has lead to an 

improvement in DNA collection swabs (Marshall et al., 2014), we are optimistic that 

results from our study and other recent studies about DNA loss (Lee et al., 2010; 

Dabney et al., 2013; Barta et al., 2014b) will encourage researchers to focus attention on 

potentially solving, or at least minimizing the problem associated with DNA extraction, as 

was recently done by Dabney and colleagues (2013). It would be very interesting to see 

if their modified protocol is also useful in retaining lower copy number standards, since

that would, presumably, be a more accurate reflection of starting copy numbers in 

degraded, ancient and/or LCN samples. 

During the present study, individual average loss of amplicons ranged from 21.75% to 

60.56% (mean 39.03%), which is lower than that observed (~71-74%) of a single 181 bp 

standard using the Promega Wizard® PCR Preps Purification System and the QIAquick 

PCR purification kit (Barta et al., 2014b). This might be the result of carrier effect within 

a pooled standard of 16 different sized fragments. It could also be a product of using the 

Qiagen MinElute PCR Purification Kit, which employs a different chemistry compared to 

the Promega Wizard® PCR Preps Purification System, and uses a modified fixed silica

column compared to that employed in the QIAquick PCR purification kit.

Our results are inconsistent with Qiagen’s (2008) claim that 80% of DNA fragments 

ranging 70 bp to 4 kb in length are retained (i.e., only a 20% loss) by the MinElute PCR 

Purification Kit. This might be a product of our deviations from the published protocol 

(Qiagen, 2008). First, we did not add pH indicator to the Buffer PB to determine if the 

mixture of this buffer with our DNA standards resulted in suboptimal pH [i.e., indicated 

when the Buffer PB (with pH indicator) turns from yellow to orange or violet]. 

Subsequently, we tested whether mixtures of 1 ×	107, 1 ×	105, and 1 ×	104 pooled 

standards and the Buffer PB (with pH indicator) resulted in suboptimal pH; they did not. 

Secondly, we eluted DNA in the final step with 10mM Tris-HCl, pH 8.5 instead of using 

Buffer EB. Yet, as described in the Qiagen (2008) manual and at their website 

(www.qiagen.com), Buffer EB is 10mM Tris-HCl, pH 8.5. In either case, we do not feel 

that these deviations from protocol explain much, if any, of the deviation between 

Qiagen’s claim of 20% loss and our observed loss of 21.75% to 60.56% (mean 39.03%). 

Since Qiagen (2008) does not report on the variance of their observed loss, it is 

impossible to know if it overlaps sufficiently with ours to warrant no statistical difference 

between our observed means and theirs.

As highlighted by Barta and colleagues (2014b), manufacturers’ methods that lead to 

claims of extraction efficiencies are typically not described, which is true for the Qiagen 

MinElute PCR Purification Kit (Qiagen, 2008). While the Qiagen (2008) manual 

describes visual estimation of DNA loss of a 5.5 kb fragment on an agarose gel, it is not 

clear how this relates to their estimation of the efficiency of the MinElute PCR 

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necessarily reflect the official position or policies of the U.S. Department of Justice.

20
 

Purification Kit. It should be the responsibility of manufacturers to clearly and more 

accurately describe how their observations were made and/or to produce peer reviewed 

reports that could be scrutinized by members of the scientific community.

On a related note to manufacturers’ claims, it is incredible that Dabney and colleagues 

(2013) were able to retain ~95% of 35 bp fragments (estimated from their Fig 1), given 

Qiagen’s (2008) claims that the Qiagen MinElute PCR Purification Kit will specifically 

remove fragments ≤40 bps. Further understanding of what specific aspect of their 

modified protocol led to this unexpected recovery is needed, because at present the 

cause of this effect is not clear (i.e., buffer composition or volume, or perhaps both 

modifications are required). 

Our experiments to evaluate the relationship between DNA strand length and retention, 

and also that of starting copy number and retention, revealed no clear linear 

relationships. With regard to the former, DNA binding efficiency to silica should be 

unrelated to molecule length (Melzak et al., 1996), yet silica methods are used 

specifically to remove short fragments of DNA (e.g., Qiagen, 2008) and have been 

demonstrated empirically to do so, at least with the method of Rohland and Hofreiter 

(2007a) conducted by Dabney and colleagues (2013). It is interesting that we observed 

no relationship between starting concentration and DNA loss, which suggests that we

did not reach a saturation point of DNA on the silica column. Yet this saturation point

does not appear to have been reached even by Dabney and colleagues (2013) with a 

much higher copy number standard (1 ×	1010). This begs an important question 

regarding the mechanism of DNA loss that has yet to be addressed—is the DNA not

binding efficiently to the silica, or is it not being efficiently released from the silica once it 

is bound? Additional experiments to resolve this question could lead to some intriguing 

insights. 

Models are, by their nature, inaccurate representations of reality, built to be simple, and 

to test specific aspects of reality. We are well aware that our “naked” DNA standards 

mimic only the sizes, and possible concentrations, of DNA typically recovered from 

degraded and ancient samples. It would be ideal to be able to generate synthetic DNA 

standards that exhibit, for example, a known degree of cytosine deamination and/or 

crosslinking to other biomolecules (e.g., that which forms Maillard products), and/or are 

in association with known quantities of PCR inhibitors. While some of these associated 

variables could lead to better retention of DNA during the extraction and purification

processes, the mechanism(s) by which they would work are presently not clear. The 

experiments of Lee and colleagues (2010) demonstrate that the efficiency of silica based 

extraction in retaining 50 ng of genomic DNA is largely unaffected by the presence of

hematin (12-60 nmol) or humic acid (1.5-15.0 μg). However, simply mixing some 

quantity of DNA with some concentration of PCR inhibitors may also not be a good 

reflection of reality. For example, even with the potential of losing a tremendous amount 

of DNA each time they are conducted, repeated silica extractions have proven very 

useful in the retrieval of DNA from ancient specimens associated with high amounts of 

PCR inhibitors (Kemp et al., 2006; Grier et al., 2013; Kemp et al., 2014; Moss et al., 

2014). 

These observations make it obvious to us that reality is more complex than any model 

being proposed. Others have noted this as well. For example, in recognition that 

previous assessments of PreCRTM DNA repair treatment were conducted on naked DNA 

controls, Ambers and colleagues (2014b) sought to determined how well this treatment 

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Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

21
 

would repair experimentally degraded “native” DNA. Interestingly, they first noted that 

damaging native nuclear DNA was more difficult than damaging naked DNA, 

presumably because the former is found tightly coiled and complexed to various proteins 

within the cell nucleus. Secondly, they demonstrated that DNA repair was more efficient 

on bleach-treated bloodstains in comparison to environmentally exposed bloodstains 

and bone samples. Following the rationale of Ambers and colleagues (2014b), it is 

important to draw caution over the experimental results reported here based on naked

DNA. It is possible that degraded native DNA will behave drastically different when 

subjected to the extraction method tested here. However, it is currently impossible to 

determine how much DNA is present in a degraded bone sample without first extracting 

and purifying to its naked state. That is, one cannot determine how much DNA is 

present in a bone sample, only the amount that is retained following extraction and 

purification (Barta et al., 2014b), which according to our experiments using naked DNA 

standards could be associated with tremendous loss of genetic material. Moreover, 

DNA preserved across single elements may be too variable to assume that one piece of 

a bone will contain equivalent DNA copy number to another piece (Barta et al., 2014a).

Future experiments could be designed to investigate the behavior of damaged native 

DNA across various extraction and purification methods.

Given that there are about as many extraction protocols as there are labs working with 

aDNA (Anderung et al., 2008), we do not claim to know the best method for reducing

loss. We have only tested one such protocol here and previously we evaluated a few 

others (Barta et al., 2014b). However, researchers using any method can adopt the 

rationale and methodological outline we provide for testing DNA loss against a standard. 

We strongly encourage others to follow our lead [or similar approach (Lee et al., 2010;

Dabney et al., 2013)]. By comparing extraction results against a standard, testing the 

efficiency of DNA retention of any and every method is possible. 

Twenty-five years following initial demonstrations that ancient bones contain preserved 

DNA, even hundreds to thousands of years after death, we are still trying to resolve the 

many difficulties and unique obstacles generated by the study of degraded and LCN 

DNA. The power to derive even partial profiles from skeletal elements is indispensable 

to the forensic sciences and the aDNA field. However, it is advisable to remain humble 

to the notion that there are still fundamental aspects of DNA preservation and its 

extraction that are poorly understood. Our study and those of others now collectively 

suggest that there may be appreciably more DNA preserved in ancient and degraded 

bone samples, and demonstrate that the mechanisms for retaining DNA in extracts may 

be highly variable. Any additional amount of DNA that can be retained through the 

extraction process would only serve to improve the ability to close forensic cases and 

develop more accurate reconstructions of the evolutionary history of humans and other 

organisms. 

ACKNOWLEDGMENTS 

Thanks to Kathleen Judd for assistance in the laboratory. 

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Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

22
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Poinar H (2009) New insights from old bones: DNA preservation and degradation 

in permafrost preserved mammoth remains. Nucleic Acids Res 37:3215-29. 

Stoneking M, Hedgecock D, Higuchi RG, Vigilant L, and Erlich HA (1991) Population 

variation of human mtDNA control region sequences detected by enzymatic 

amplification and sequence-specific oligonucleotide probes. Am J Hum Genet 

48:370-82. 

van Oorschot RA, Ballantyne KN, and Mitchell RJ (2010) Forensic trace DNA: a review. 

Investig Genet 1:14. 

van Oorschot RAH, Phelan DG, Furlong S, Scarfo GM, Holding NL, and Cummins MJ 

(2003) Are you collecting all the available DNA from touched objects? 

International Congress Series 1239:803–807. 

Warinner C, Rodrigues JF, Vyas R, Trachsel C, Shved N, Grossmann J, Radini A, 

Hancock Y, Tito RY, Fiddyment S, Speller C, Hendy J, Charlton S, Luder HU, 

Salazar-Garcia DC, Eppler E, Seiler R, Hansen LH, Castruita JA, BarkowOesterreicher S, Teoh KY, Kelstrup CD, Olsen JV, Nanni P, Kawai T, Willerslev 

E, von Mering C, Lewis CM, Jr., Collins MJ, Gilbert MT, Ruhli F, and Cappellini E 

(2014) Pathogens and host immunity in the ancient human oral cavity. Nat Genet 

46:336-44. 

Winters M, Barta JL, Monroe C, and Kemp BM (2011) To clone or not to clone: Method 

analysis for retrieving consensus sequences in ancient DNA samples. PLoS One 

6:e21247. 

Yang DY, Eng B, Waye JS, Dudar JC, and Saunders SR (1998) Technical note: 

Improved DNA extraction from ancient bones using silica-based spin columns. 

American Journal of Physical Anthropology 105:539-543. 

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28
TABLE 1. Primers used in the PowerPlex 16® System, genotype and ampicon lengths of Promega 9948 M

sequences taken from Krenke et al. (2002) and Male 9948 genotypes are as reported in the PowerPlex 16® Sys

manual, from which amplicon lengths were calculated.

Promega 9948 Male Individual qPCR 

DNA Reactions 

Amplicon

Locus Primer Sequence 5' to 3' Genotype Sizes (bps) Efficiency (%) R2 Ef

Amelogenin CCCTGGGCTCTGTAAAGAA ATCAGAGCTTAAACTGGGAAGCTG X, Y 106, 112 96.85 0.9991 

D3S1358 ACTGCAGTCCAATCTGGGT ATGAAATCAACAGAGGCTTGC 15, 17 127, 135 95.03 0.9981 

D5S818 GGTGATTTTCCTCTTTGGTATCC AGCCACAGTTTACAACATTTGTATCT 11, 13 135, 143 95.60 0.9981 

vWA GCCCTAGTGGATGATAAGAATAATCAGTATGTG GGACAGATGATAAATACATAGGATGGATGG 17, 17 151 104.12 0.9979 

TH01 GTGATTCCCATTGGCCTGTTC ATTCCTGTGGGCTGAAAAGCTC 6, 9.3 164, 184 93.08 0.9967 

D13S317 ATTACAGAAGTCTGGGATGTGGAGGA GGCAGCCCAAAAAGACAGA 11, 11 192 92.29 0.9994 

D21S11 ATATGTGAGTCAATTCCCCAAG TGTATTAGTCAATGTTCTCCAGAGAC 29, 30 223, 227 92.75 89.41† 0.9966 0.9989† 

D8S1179 ATTGCAACTTATATGTATTTTTGTATTTCATG ACCAAATTGTGTTCATGAGTATAGTTTC 12, 13 223, 227 97.74 0.9961 

D7S820 ATGTTGGTCAGGCTGACTATG GATTCCACATTTATCCTCATTGAC 11, 11 235 90.66 0.9988 

TPOX GCACAGAACAGGCACTTAGG CGCTCAAACGTGAGGTTG 8, 9 270, 274 98.82 0.9953 

D16S539 GGGGGTCTAAGAGCTTGTAAAAAG GTTTGTGTGTGCATCTGTAAGCATGTATC 11, 11 288 96.77 0.9989 

D18S51 TTCTTGAGCCCAGAAGGTTA ATTCTACCAGCAACAACACAAATAAAC 15, 18 318, 330 91.08 86.79§ 0.9929 0.9962§ 

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CSF1PO CCGGAGGTAAAGGTGTCTTAAAGT ATTTCCTGTGTCAGACCCTGTT 10, 11, 12 337, 341, 345 91.10 0.9992 

FGA GGCTGCAGGGCATAACATTA ATTCTATGACTTTGCGCTTCAGGA 24, 26 354, 362 98.68 0.9965 

Penta E ATTACCAACATGAAAGGGTACCAATA TGGGTTATTAATTGAGAAAACTCCTTACAATTT 11, 11 409

77.25

87.19§

75.81†

0.9960

0.9957§

0.9983†

614.3 0.0876

Penta D GAAGGTCGAAGCTGAAGTG ATTAGAATTCTTTAATCTGGACACAAG 8, 12 408, 428 42.00§ 49.74* 0.9350§ 0.9871* 

68.05† 0.9919†

* from a second dilution series created from the original amplification † from a second set of amplifications and dilution series created from those reactions§ repeat qPCR from original dilutions

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TABLE 2. Summary of percent DNA loss across all fifteen markers evaluated from three standards. See 

details.

Percentage loss of 1

× 104 standard Percentage loss of 1

× 105 standard Percenta

Locus Size* 1 2 3 4 Mean SD 1 2 3 4 Mean SD 1 2 

Amelogenin 109 60.21 62.99 58.54 60.51 60.56 1.84 59.70 48.25 55.20 53.96 54.28 4.72 43.11 39.3

D3s1358 131 31.72 44.92 54.04 50.79 45.37 9.85 48.76 47.02 49.78 51.49 49.26 1.87 29.09 27.2

D5s818 139 25.35 34.74 57.99 56.38 43.61 16.14 42.62 54.18 47.42 50.05 48.57 4.84 31.62 26.9

vWA 151 25.90 22.99 37.70 40.41 31.75 8.59 27.54 25.64 31.81 30.84 28.96 2.87 41.78 22.7

TH01 174 43.08 36.30 44.95 46.40 42.68 4.47 46.56 39.27 37.04 42.40 41.32 4.13 46.55 32.8

D13s317 192 49.52 49.17 51.51 45.93 49.03 2.31 53.00 39.30 37.61 38.76 42.17 7.26 28.40 32.1

D21s11 225 49.45 45.66 42.97 32.96 42.76 7.05 42.41 33.75 32.80 30.35 34.83 5.25 27.30 25.4

D8s1179 225 36.06 46.11 40.68 45.02 41.97 4.58 39.83 39.06 35.05 29.11 35.76 4.90 21.15 25.4

D7s820 235 29.33 48.16 48.95 46.98 43.35 9.39 37.63 35.38 40.91 34.91 37.21 2.74 39.58 36.5

TPOX 272 34.56 25.32 30.18 39.92 32.49 6.22 50.57 42.70 30.11 42.20 41.39 8.44 34.62 27.2

D16s539 288 44.96 45.14 41.97 42.89 43.74 1.56 47.26 34.60 39.70 40.00 40.39 5.21 32.17 29.6

D18s51 324 51.65 56.36 47.62 35.67 47.82 8.85 50.70 50.80 38.09 36.73 44.08 7.72 39.26 40.7

CSF1PO 341 48.79 49.30 45.38 42.64 46.53 3.12 48.30 47.64 36.59 37.79 42.58 6.25 31.81 32.2

FGA 358 27.15 33.49 46.60 44.40 37.91 9.18 40.30 53.52 42.36 40.58 44.19 6.28 27.35 27.6

Penta E 409 33.93 41.76 29.24 33.80 34.68 5.20 41.61 38.89 43.90 38.63 40.76 2.49 36.49 36.6

Average loss: 42.95 7.17 Average loss: 41.72 6.23 

*Average amplicon size based on genotype of Promega Male 9948 (Table 1)

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Figure 1. Regression of average percent DNA loss against DNA fragment 

size, taken from data presented in Table 2. Black diamonds represent 

standards at 104, dark gray squares represent standards at 105, and light gray 

triangles represent standards at 107. R squared and p-values for each slope are 

indicated. 

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32
Appendix A. Quantitative PCR results based on 104 standards. These data are 

summarized in Table 2. 

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33
Appendix A (Continued). Quantitative PCR results based on 105 standards. . 

These data are summarized in Table 2. 

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34
Appendix A (Continued). Quantitative PCR results based on 107 standards. . 

These data are summarized in Table 2. 

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35
 

Chapter 2: Are We Fishing or Catching? Evaluating the 

Efficiency of Bait Capture of CODIS Fragments 

Misa Wintersa,b, Cara Monroea, Jodi Lynn Bartac, and Brian M. Kempa,b

a Department of Anthropology, Washington State University, Pullman, WA 99164-4236 

b School of Biological Sciences, Washington State University, Pullman, WA 99164-4910 c Department of Biological and Health Sciences, Madonna University, Livonia, MI 48150

Corresponding Author: 

Brian M. Kemp 

Department of Anthropology 

Washington State University 

Pullman, WA 99164 

Office: 509-335-7403 

Fax: 509-335-3999 

bmkemp@wsu.edu

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36
ABSTRACT

This study sought to document the efficiency of DNA bait capture (i.e., “fishing”) methods 

by two measures: 1) its ability to retain targeted DNA molecules, and 2) its ability to 

remove non-target DNA molecules from a pool containing both. Efficiencies were 

estimated by comparing the number of “copies in” to “copies out” with quantitative 

polymerase chain reaction (qPCR). Retention of target DNA molecules, ranging 109-

288 base pairs (bps) in length, averaged just 9.06-3.53% (i.e., loss of 90.94-96.47%) 

using the fishing protocol as previously described. Some improvement was achieved by 

employing a modified protocol (i.e., with a shortened hybridization time, use of twice the 

amount of M-270 streptavidin-coated beads, and modified bead washing), resulting in

average retention of 31.41-12.08% of the same set of targeted molecules. Noted was 

the lack of efficacy in removing non-target DNA molecules as opposed to targeted 

molecules. It was also observed that most of the molecules (61.35-69.49%) are “lost”

during the essential hybridization step of the fishing protocol, suggesting its suitability for 

high copy number samples only. While the bait capture method may be useful in the

study of DNA compromised by polymerase chain reaction (PCR) inhibitors as previously 

suggested, it is necessary to carefully weigh this possible advantage against the degree 

of expected DNA loss and the non-selectivity of the method for targeted over nontargeted DNA. 

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37
INTRODUCTION 

The analysis of DNA extracted and purified from low copy number (LCN), ancient, and/or 

degraded source materials is largely complicated by: 1) the presence of contaminating 

“modern” (Kemp and Smith, 2005; Barta et al., 2014) and “ancient” (Noonan et al., 2005; 

Poinar et al., 2006) DNA, 2) co-extracted polymerase chain reaction (PCR) inhibitors 

(Alaeddini, 2011; Kemp et al., 2014a), and 3) the degree to which template molecules 

have been damaged or chemically modified post-mortem or from the time of deposition 

of the biological material (Gilbert, 2006; Alaeddini et al., 2010). As these associated 

problems make the authentication of DNA profiles from such samples particularly

problematic, there is continued need for the development and evaluation of methods that 

increase the yield and purity of genetic material extracted from degraded sources.

Anderung et al (2008) developed a method of “fishing” for DNA, building on the “targethooking” method described by Tofanelli and Nencioni (1999). The method works by 

denaturing DNA and hybridizing target molecules to synthetic biotinylated DNA primers 

(i.e., “probes” or “bait”) (Figure 1). Immobilization of this hybridized complex onto 

streptavidin coated magnetic beads effectively “captures” the targets. Application of a 

strong magnet attracts the DNA-bound beads to the side or bottom of the tube while 

buffers are used to wash away impurities and non-target DNA. Once washed, the beads 

are released from the magnet and targeted DNA is disassociated from the probes in 

preparation for downstream applications. In direct comparison to a more widelypracticed silica-based extraction method (Yang et al., 1998), Anderung et al (2008) 

found the performance of the fishing method to be at least equivalent in retrieving DNA 

originating from ancient cattle bones and teeth (ranging in age from the Neolithic to the 

Mediaeval period). It is notable, however, that for two of twelve samples they analyzed, 

only the fishing method yielded amplifiable DNA. Anderung et al (2008) argued that 

fishing for DNA should produce volumes of template DNA that are free of PCR inhibitors. 

They also suggested, but did not demonstrate, that serial rounds of fishing for DNA 

could be used to concentrate molecules of interest from a pool of target and non-target 

molecules. At a minimum, Anderung et al (2008) were the first to demonstrate the 

successful use of a capture-based method directly on degraded, LCN extracts. 

With the goal of disentangling mixtures of human and non-human DNA, Nandineni and 

Vedanayagam (2009) used a fishing approach to capture Combined DNA Index System 

(CODIS) short tandem repeats (STRs), plus the D2s1338, D19s433, and Amelogenin 

markers. In total, these are the markers genotyped by the AmpFlSTR® Identifiler® PCR 

Amplification Kit (Life TechnologiesTM). They designed 16 biotinylated primers that 

probe a few nucleotides upstream from where the proprietary AmpF/STR® primers 

anneal (Collins et al., 2004), and captured these markers from human DNA that had 

been sonically fragmented to resemble the state of typically degraded DNA samples. 

Successful genotyping was achieved for all loci, albeit on an unreported number of 

samples. Furthermore, Nandineni and Vedanayagam (2009) noted, but did not describe 

in any detail, that capture was unsuccessfully performed with non-biotinylated primers. 

Since hybridization to biotinylated probes is required for immobilization (Figure 1), this 

was an expected experimental result. It is peculiar that Nandineni and Vedanayagam 

(2009) did not assess the efficiency of capturing human DNA from a pool that contains

non-human DNA, as that was one of the stated foci of their study. 

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38
Building directly off the study of Nandineni and Vedanayagam (2009), Wang and 

McCord (2011) demonstrated that allelic drop out could be minimized, and low intensity 

amplification of large-fragment STRs improved, from experimentally degraded DNA 

samples after fishing with biotinylated primers. Interestingly, while determining that 

fishing for DNA would result in the removal of PCR inhibitors, they also found that the 

presence of hematin at 0.8 mM or humic acid at 0.24 mg/mL (i.e., PCR inhibitors) might 

actually interfere with the essential hybridization step.

None of the studies reviewed above documented the efficiency of fishing for target DNA 

or that of non-target DNA removal by measuring against a standard of known 

concentration. In the case of the Anderung et al (2008) study, comparison to the silicabased method of Yang et al. (1998) is useful, but the comparison is a relative one; 

neither method was tested for its efficiency against a known quantity of DNA. 

Subsequently, it has been demonstrated that the Qiaquick PCR Purification kit, as 

employed by Yang et al. (1998), is associated with the loss of 71.25% (SD 30.56%) of a 

known standard containing naked DNA fragments 181 base pairs (bps) in length (Barta 

et al., 2014). Yet, measured efficiency against a known quantity of DNA has not been 

established for the fishing method. Likewise, the efficiency of capturing CODIS markers 

against a standard of known quantity was not established by either Nandineni and 

Vedanayagam (2009) or Wang and McCord (2011). While the latter study compared the 

results relative to genotyping from a pool of degraded, non-captured DNA, this simply 

demonstrated that the method works, not how efficient it is.

In this study, we sought to document the efficiency of fishing for CODIS STRs sized 

fragments following the approach of comparing “copies in” to “copies out”, as estimated 

by quantitative PCR (qPCR) (Barta et al., 2014; Kemp et al., 2014b) (Chapter 1). This 

general approach allowed us to measure efficiency of capturing target DNA molecules 

while simultaneously measuring the efficiency of removing non-target DNA molecules 

from a common pool containing both (Figure 2).

MATERIALS AND METHODS

Creation of the Pooled Standards 

The Promega PowerPlex 16® System was chosen to create DNA standards because it 

targets the thirteen CODIS STRs, in addition to the amelogenin, Penta D, and Penta E 

markers. The amplicons produced from the Promega 9948 Male DNA sample range in 

size from 106 bps of the amelogenin gene on the X chromosome to 428 bps from the 

Penta D locus on chromosome 15 (Kemp et al., 2014b) (Chapter 1). Critical to our 

experimental design choice were the published PowerPlex 16® System primer 

sequences and casework validations (Masibay et al., 2000; Krenke et al., 2002; Butler et 

al., 2003), as knowing the primer sequences was essential for constructing the 

standards, as described below. 

Each of the sixteen fragments of the human genome targeted by PowerPlex 16® System

were individually amplified eight times in 30 μL PCRs containing: 0.32 mM dNTPs, 1X

PCR buffer, 1.5 mM MgCl2, 0.24 μL primers (described by Krenke et al., 2002), 0.6 U 

Platinum Taq DNA Polymerase (Life Technologies), and 1.5 μL of Promega 9948 Male 

(10 ng/μL) DNA. These were used as target DNA in the capture experiments described 

below. PCR negatives accompanied these reactions to monitor for contamination. 

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39
Cycling was performed with an initial 3 minute hold at 94°C followed by 40 cycles of 15 

second holds at 94°C, 60°C, and 72°C, followed by a 3 minute hold at 72°C. 

A 181 base pair (bp) portion of the northern fur seal (Callorhinus ursinus) mitochondrial 

cytochrome B gene was PCR amplified eight times using primers NFS-F5-CtB and NFSR185-CtB (Moss et al., 2006). This was used as non-target DNA in the capture 

experiments described below. These amplicons will henceforth be referred to as “NFS 

mtDNA”. Each 30 μL PCR contained: 0.32 mM dNTPs, 1X PCR buffer, 1.5 mM MgCl2, 

0.24 M primers, 0.6 U Platinum Taq DNA Polymerase, and 1.5 μL of northern fur seal 

DNA. PCR negatives accompanied these reactions to monitor for contamination. 

Cycling was performed with an initial 3 minute hold at 94°C followed by 40 cycles of 15 

second holds at 94°C, 55°C, and 72°C, followed by a 3 minute hold at 72°C. 

Successful amplification was confirmed by separating 4 μL of PCR products using 2%

agarose gel electrophoresis, and visualizing with ethidium bromide staining under 

ultraviolet (UV) illumination. The remaining volumes of each set of eight reactions were 

pooled and purified with the Qiagen QIAquick PCR Purification Kit following the 

manufacturer’s protocol except that the pH indicator was not added and the final elution 

was conducted with molecular grade water. Following purification of the amplicons, 

standard concentration was determined by taking the average of 2-3 spectrophotometry 

readings using a Nanodrop (Thermoscientific), from which copy numbers were 

calculated as follows (Kemp et al., 2014b) (Chapter 1): 

number of copies = ([amount (ng)] ×	[6.022 ×	1023])/ ([length (bp)] × [1 ×	109] × [650])

The seventeen amplicons were pooled and diluted to final concentrations of 1 ×	105, 1 ×	

104, and 1 ×	103 copies/μL. For example, the 1 ×	105 dilution contained 100,000 copies 

of each of the sixteen PowerPlex 16® System amplicons and 100,000 copies of northern 

fur seal mtDNA per μL.

Indirect Fishing Protocol (largely following Anderung et al., 2008) (Figure 1) 

This fishing protocol is referred to as “indirect”, in contrast to what we describe as the 

“direct” method below, because this protocol hybridizes the target DNA to the 

biotinylated primers (through the remainder of this paper, these will be referred to as 

“probes”) and then immobilizes the hybridized product to the streptavidin-coated beads. 

In contrast, the “direct” method employs immobilization of the probes to the streptavidincoated beads prior to hybridization of the target DNA (Figure 3). 

Sample hybridization 

A 50 μL volume of the pooled standard was added to 1000 μL of 1X Binding and Wash 

(B&W) buffer (5 mM Tris-HCl ph 7.5, 0.5 mM EDTA, 1 M NaCl) and then mixed. Ten μL 

of PowerPlex 16® System biotinylated probes (at a concentration of 0.005 pmol/μL each) 

were added to each tube and mixed. Tubes were incubated at 100°C for 15 minutes, 

immediately placed on ice, transferred to a -20°C freezer for 15 minutes, and incubated 

at 50°C for 30 minutes. Tubes were then allowed to return to room temperature. 

Bead Preparation 

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Forty microliters of Dynabeads® M-280 Streptavidin-coated beads were prepared by first 

adding 1000 μL of 1X B&W buffer. This solution was vortexed and placed on the 

magnet (DynamagTM) for 2 min, followed by discarding the supernatant. An additional 

200 μL volume of 1X B&W buffer was added, the tube vortexed, and placed on the 

magnet for 2 minutes, followed by discarding the supernatant. This was repeated two 

additional times for a total of three washes with 200 μL of 1X B&W. A final addition of 

600 μL of 2X B&W buffer brought the concentration of the beads to 0.067 mg/mL.

Immobilization 

A 60 μL volume of the prepared bead solution (containing a total of 0.04 mg beads) was 

added to the 1060 μL of the hybridized solution, resulting in a solution containing 1X

B&W buffer. This results in a 1M NaCl concentration, the optimal binding condition, as 

described by Invitrogen (2012). This mixture was gently rocked for 30 min at room 

temperature. The samples were then placed on the magnet for 2 min, followed by 

discarding the supernatant. The beads were twice washed with 200 μL of 1X B&W 

buffer as described above, followed by two washes with PCR buffer (10mM Tris-HCl pH 

8.0, 50mM KCl). Finally, the immobilized template was re-suspended in 40 μL of dH2O. 

Modified Indirect Fishing Protocol #1 (M-270 beads) 

The indirect fishing protocol described above was followed with the substitution of 

Dynabeads® M-270 streptavidin-coated beads for M-280 beads. 

Modified Indirect Fishing Protocol #2 (modified hybridization, 2X M-270 beads, 

modified bead washing) 

The indirect fishing protocol described above was executed with the following 

modifications: 1) during hybridization, tubes were incubated at 100°C for 15 minutes, 

immediately placed on ice for 15 minutes, then allowed to return to room temperature 

(i.e., bypassing the 30 min hold at 50°C), 2) twice the amount of Dynabeads® M-270 

streptavidin-coated beads were substituted for M-280 beads, and 3) following 

immobilization, the beads were thrice washed with 200 μL of 1X B&W buffer, followed by 

resuspension in 50 μL of dH2O.

Direct Fishing Protocol (Figure 3) 

Bead Preparation and Immobilization of Primers 

Eighty microliters of Dynabeads® M-270 Streptavidin-coated beads were prepared as 

described above for the indirect fishing protocol. The final elution with 600 μL of 2X

B&W buffer resulted in the final concentration of beads at 0.134 mg/mL.

To 60 μL of prepared beads was added a 10 μL volume of PowerPlex 16® System 

biotinylated probes (at a concentration of 0.005 pmol/μL each) and the tubes were gently 

shaken for 30 min at room temperature. 

Sample hybridization 

A volume of 50 μL of pooled standard was added to the tubes containing beads and 

probes. This resulted in 120 μL of bead-immobilized beads in a 1 X B&W buffer. Tubes 

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41
were incubated at 100°C for 15 minutes, placed on ice 15 minutes, and allowed to return 

to room temperature. The tubes were placed on the magnet for 2 min and the 

supernatant removed. The beads were thrice washed with 200 μL of 1X B&W buffer and 

finally resuspended in 50 μL of dH2O.

Hybridization Only (from Indirect Fishing Protocol) 

The effect of hybridization alone (following sample hybridization described under the 

indirect fishing protocol and depicted as steps 1-2 in Figure 1) on the DNA standards 

was assessed. Since B&W buffer inhibits qPCR, the final products had to be purified 

prior to quantification (described below).

A volume of 1000 μL of 1X B&W buffer was added to 50 μL of the pooled standard, 

along with 10 μL of PowerPlex 16® System biotinylated probes (at a concentration of 

0.005 pmol/μL each) and subsequently mixed. Tubes were then incubated at 100°C for 

15 minutes, immediately placed on ice, and incubated at 50°C for 30 minutes. Tubes 

were then allowed to return to room temperature. 

Aliquots of 132.5 μL of the hybridized solutions were purified using the Qiagen MinElute 

PCR Purification Kit. The manufacturer’s protocol was followed except that 132.5 μL of 

10mM Tris-HCl, ph 8.5 was added to the center of the column membrane and left at 

room temperature for 1 min prior to centrifugation into clean 1.5 mL tubes. As this 

volume of DNA is 1/8 the initial concentration (i.e., 132.5/1000), final values from qPCR 

results were corrected by multiplying by eight. 

Modified Hybridization Only (from Modified Indirect Fishing Protocol #2) 

To 50 μL of the pooled standard was added a 1000 μL volume of 1 X B&W buffer and 10 

μL volume of PowerPlex 16® System biotinylated primers (at a concentration of 0.005 

pmol/μL each) and the tubes mixed. Tubes were incubated at 100°C for 15 minutes and 

immediately placed on ice. Tubes were then allowed to return to room temperature. 

Samples were purified with the Qiagen MinElute PCR Purification Kit and final values 

adjusted as described above. 

Quantitative PCR (qPCR) and Calculating Percent Retention and Loss 

The amount of amelogenin, D13s317, D16s539 DNA remaining in the standard following 

capture with the various fishing protocols described above or following speficial 

subjection to the hybridization steps (i.e., “copies out”) was determined by qPCR 

following Kemp et al. (2014b) (Chapter 1). Quantitative PCR reactions of 25 μL 

contained: 12.5 μL SYBR Green Real-Time PCR Master Mix (Life Technologies), 0.5 

mM Rox, 0.4 μM of each primer, and 5 μL of template DNA. Cycling conditions were as 

follows: 50°C for 2 min, 95°C for 3 min, followed by 50 cycles of holds at 95°C for 15 

sec, 60°C for 30 sec, 72°C for 30 sec, and then a dissociation step of 95°C for 15 sec 

and 60°C for 1 min. 

These three markers were chosen as representatives of the 16 fragments targeted 

during hybridization. The average Amelogenin, D13s317, D16s539 fragment lengths are 

109, 192, and 288 bps. Moreover, the efficiencies (E, 96.07-100.81%) and coefficient of 

determination (R2, 0.9970-0.9982) of these reactions were previously determined to be 

acceptable when screened in a pool (Kemp et al., 2014b) (Chapter 1). 

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42
 

The amount of NFS mtDNA remaining in the standards was also determined via qPCR. 

This represents non-target DNA retained following subjection to capture or hybridization 

only. Quantitative PCR reactions were conducted as described above. 

Quantification of each marker was conducted in duplicate against standard curves 

generated from two reactions each of standard pools at 1 ×	105, 1 ×	104, 5 ×	103, 2 ×	103, 

1 ×	103, 5 ×	102, 2.5 ×	102, 1 ×	102 and 50 copies/μL. Two additional wells of each 

untreated 1 ×	105, 1 ×	104, and 1 ×	103 copies/μL standards were quantified as 

unknowns. These provided estimates of “copies in”.

Subtraction of the number of “copies out” (measured as the average of duplicate qPCR 

amplifications) from “copies in” (measured as the average of duplicate qPCR 

amplifications of the standards treated as unknowns) divided by “copies in” multiplied by 

100 provides the percent retention:

[(copies in - copies out)/ copies in] ×	100 

One hundred minus retention provides a measure of percent loss. Retention and loss of 

targeted amelogenin, D13s317, D16s539, and non-targeted NFS mtDNA was 

determined in this manner three times, from which the average loss and the uncorrected 

sample standard deviation (i.e., the standard deviation of the sample) were calculated. 

Statistical Analyses 

Two-tailed t-tests were used to statistically evaluate differences in mean percentage 

retention at the 0.05 level of probability. Statistical analyses were conducted in StatPlus 

(AnalystSoft Inc.).

RESULTS 

Quantitative PCR efficiencies for amelogenin, D13s317, and D16s539 ranged from 

91.33-98.91%, with R2 values ranging 0.9983-0.9996 (Tables 1-6), which are 

comparable values to those previously observed (Kemp et al., 2014b) (Chapter 1). The 

NSF mtDNA qPCR efficiencies ranged from 90.20-92.96% with R2 values from 0.9963-

0.9994. 

Across all three standards (1 × 105, 1 ×	104, and 1 ×	103 copies/μL), average DNA 

retention of the targeted amelogenin, D13s317, and D16s539 by the indirect fishing 

method ranged from 9.06-3.53% (i.e., loss of 90.94-96.47%; see Table 1). The retention 

of the non-targeted NFS mtDNA was similar at 4.02-6.64% (two-tailed t-test, p=0.633). 

Substitution of M-270 streptavidin-coated beads for M-280 beads resulted in an average 

retention of 13.33-7.82% for amelogenin, D13s317, D16s539, and 17.43-10.10% for 

NFS mtDNA (Table 2). The mean difference between percent retention of the targeted 

markers was not statistically different from that of the non-targeted NSF mtDNA 

(p=0.102). The second modified indirect fishing protocol (i.e., modified hybridization, 2X

M-270 beads, modified bead washing) resulted in average retention of 31.41-12.08% for 

amelogenin, D13s317, D16s539, which was not statistically different from the 24.65-

22.07% retention of the NFS mtDNA (p=0.623) (Table 3). The average retention for the 

three targeted markers by the direct fishing method (0.16-0.02%) was not statistically 

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43
different from that observed for the non-targeted NFS mtDNA (0.25-0.12%, p=0.129) 

(Table 4). 

The mean retention of targeted molecules by the second modified indirect method 

(21.70%,Table 3) was larger than that of the means of the indirect method (5.79%, p=2.4 

×	10-7, Table 1), the first modified indirect method (11.37%, p=5.3 ×	10-5, Table 2), and 

the direct method (0.10%, p=1.7 ×	10-9, Table 3). 

Standards subjected to only the hybridization step from the indirect method witnessed 

losses ranging from 75.52-90.07% (Table 5). Standards subjected only to the 

hybridization step from the second modified indirect fishing protocol resulted in a loss of 

81.01-93.08% (Table 6). The average loss of the latter (87.36%) exceeds that of the 

former (83.15%) (p=0.032). 

DISCUSSION 

In this study we sought to document the efficiency of fishing for DNA two measures, first 

in its ability to retain targeted DNA molecules and, secondly, in its ability to remove nontarget DNA molecules from a pool containing both. Following the indirect protocol of 

Anderung et al (2008) results in losses exceeding 90% across the three targeted 

markers (amelogenin, D13s317, and D16s539) (Table 1). Using the direct fishing 

method, the average loss was further exacerbated (Table 4). The average loss for direct 

fishing in comparison to the indirect fishing method was reduced with the substitution of 

M-270 streptavidin-coated beads for M-280 beads (Table 2). The difference between 

beads is that the latter are hydrophilic, whereas the former are hydrophobic (Invitrogen, 

2010). Further manipulation of the indirect fishing method, including a reduced time of 

hybridization, use of twice the amount of M-270 beads, and modified washing of the 

beads, resulted in the best retention values of target molecules at >20% (Table 3). 

While it is likely that further manipulation of the basic indirect fishing protocol could lead 

to increased efficiency of retaining targeted molecules, one must still contend with the 

fact that the method binds both target and non-target DNA rather than selectively 

hybridizing only to the target DNA molecules. Across all experimental trials, the amount 

of the non-targeted NFS mtDNA retained was statistically indistinguishable from the 

amount of targeted DNA retained (Tables 1-4). These observations clearly show that 

fishing for DNA is inefficient for both metrics considered in this study. 

Our unique approach to quantifying efficiency by comparing “copies in” to “copies out”

estimated from quantitative PCR (Barta et al., 2014; Kemp et al., 2014b) (Chapter 1) 

permitted us to begin assessing where DNA is lost in the fishing protocol. Specifically, 

by observing the effects of the hybridization conditions alone on the DNA standards, we 

noticed that over 80% of the molecules are lost in this step (Tables 5 and 6). However, 

meaningful correction can be made to that estimate. In pursuit of estimating DNA loss 

during hybridization, it was necessary to purify the DNA prior to quantification because 

the B&W Buffer inhibits PCR. Since estimates of percentage loss of Promega 

PowerPlex 16® System amplicons by Qiagen MinElute PCR Purification Kit purification 

has been established (Kemp et al., 2014b) (Chapter 1), a correction factor for loss of 

amelogenin, D13s317, and D16s539 of 52%, 40.85%, 38.95% (average loss across 

pooled standards starting at 1 ×	107, 1 ×	105, and 1 ×	104 copies/μL) can be employed. 

Once corrected, the values still demonstrate that over 60% of the molecules are lost 

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44
during hybridization (see last column in Tables 5 and 6 for corrected values). Similar 

loss of the NFS mtDNA has not been documented with the Qiagen MinElute PCR 

Purification Kit. A possible explanation for the loss following hybridization is that the 

heat treatment degrades the DNA molecules, making them non amplifiable as they no 

longer contain both priming regions, a requisite for PCR amplification. The rate of DNA 

strand breakage is related directly to temperature (Lindahl and Andersson, 1972; 

Lindahl, 1993) and it has been demonstrated that the “thermal age” of an ancient 

specimen is more meaningful in predicting DNA recovery than is calendrical age (Smith 

et al., 2003). Worthy of consideration is that in order to create degraded DNA conditions 

in their study of fishing for DNA, Wang and McCord (2011) exposed full genomic DNA in 

deionized water to 95°C temperature for 10 minutes. This implies that genomic DNA will 

readily degrade at high temperature in a short time. Admittedly, our results showing that 

hybridization conducted at 100°C for 15 minutes followed by 50°C for 30 minutes results 

in less DNA loss (Table 5) than simply heating at 100°C for 15 minutes (Table 6) is 

difficult to reconcile, given that DNA in the first treatment was exposed to heat for a 

greater period of time. Nevertheless, the magnitude of DNA loss observed in this study 

is particularly troublesome, as hybridization of targeted molecules to the biotinylated 

probes is essential to the fishing method. Even when considering this loss, one can 

conclude that, of the intact markers that are present following hybridization, only about 

half of them are retained during the best of our fishing methods. For example, if 65% of 

the molecules are lost during hybridization (estimated from Tables 5 and 6), of the 35% 

remaining, approximately 60% of them are retained (considering the observed endpoint 

average of ~21% retention, Table 3). 

If the impact of this heat treatment damage is verified by future studies, it has 

implications for improving approaches for working with degraded DNA samples. For 

example, polymerases that required extended hot starts (e.g., a 12 minute activation 

period at 95oC for Amplitaq® Gold) might be less preferable to those that require shorter 

periods of hot start (e.g., 94oC for three minutes to activate Platinum Taq). In fact, an 

informative experiment would be to compare PCR yields wherein DNA is added to the 

reactions following hot start to those wherein the DNA is added prior to the hot start 

activation. If our presumptions are correct, former will yield less DNA than the latter 

based on less template molecules available for amplification. 

It is worth emphasizing that the outcome of the experiments described here were not 

compared with standard methods of DNA extraction and genotyping, which would have 

pointed to the strength and weakness of fishing for DNA over standard methods. 

Nevertheless, as detailed in the introduction, our study is the first to evaluate the 

efficiency of fishing for DNA against 100% recovery of molecules sized exactly for 

amplification with the Promega PowerPlex 16® System. A possible future direction 

would be to experimentally degrade native DNA (e.g., Ambers et al., 2014) and evaluate 

the efficiency of fishing methods for capture of target molecules from total genomic DNA 

that is degraded and chemically modified. 

Another variable that we did not consider, but one that could form the basis for an 

informative follow-up study, is the hybridization conditions employed in our study. 

Hybridization temperatures that are too stringent (i.e., too high) would decrease the 

efficiency of targeted bait molecules to be captured. Alternatively, hybridization 

temperatures that are less stringent (i.e., too low) would permit for more non-target 

molecules to be captured. Exploration of variable hybridization temperatures and 

possibly even a touch-down hybridization step would be particularly illuminating.

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45
Fishing for DNA may still be a useful method when there is a need for purifying high 

copy number DNA associated with PCR inhibitors (Wang and McCord, 2011), however 

far less expensive and time consuming methods suitable for high and low copy samples 

have been described (Kemp et al., 2006; Kemp et al., 2014a). Therefore, the possible 

advantage of PCR inhibitor removal by fishing must be carefully weighed against the 

degree of expected DNA loss when targets are not caught, as well as the non-selectivity 

of the method for targeted over non-targeted DNA. 

ACKNOWLEDGMENTS 

Thanks to Kathleen Judd for assistance in the laboratory. 

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46
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48
Table 1. Retention of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mt

using the Indirect Fishing Protocol

Hybridization 1 Hybridization 2 Hybridization 3 Ave

E

R2 Copies In SD 

Copies

Out SD

Copies

Out SD

Copies

Out SD

Copies

Out 

Amelogenin 98.91 0.9991

129978.00 9748.87 5696.24 810.06 5726.00 1237.82 8087.27 1129.38 6503.17 137

12091.30 571.00 645.58 190.97 730.96 177.38 863.50 42.13 746.68 10

996.02 149.00 55.82 3.54 51.57 n/a 36.43 2.06 47.94 1

D13s317 93.78 0.9987

83525.20 9335.61 6999.06 n/a 7203.15 279.10 8501.16 1055.72 7567.79 81

9194.53 368.00 558.34 58.04 628.98 33.17 659.53 51.83 615.62 5

760.69 36.90 76.58 17.53 44.43 6.20 39.41 7.28 53.47 2

D16s539 96.68 0.9996

134781.00 2328.16 4748.40 825.81 5888.48 324.18 6231.40 196.67 5622.76 77

11599.60 855.00 440.30 29.62 740.86 76.29 777.91 53.10 653.02 18

1057.39 139.00 64.15 0.75 33.60 0.16 14.13 1.29 37.29 2

Average Targeted 

NFS mtDNA 92.96 0.9975

91048.80 12218.40 5878.23 n/a 5830.70 739.43 6435.91 1018.81 6048.28 33

10348.20 59.10 489.62 3.88 412.84 19.37 344.11 11.98 415.52 7

811.66 2.82 50.10 16.64 50.82 9.40 23.79 0.06 41.57 1

Average Non-Targeted 

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Table 2. Retention of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mt

using the Modified Indirect Fishing Protocol #1 (M-270 beads) 

Hybridization 1 Hybridization 2 Hybridization 3 Aver

E 

R 

2 Copies In SD 

Copies

Out SD

Copies

Out SD

Copies

Out SD

Copies

Out 

Amelogenin 96.44 0.9993

106588.00 4247.28 8862.16 641.49 6098.58 438.77 10045.44 724.19 8335.39 202

10237.80 n/a 1275.28 117.86 1014.10 39.63 1204.60 326.14 1164.66 13

1025.67 19.00 142.47 11.73 115.78 0.58 83.60 0.67 113.95 2

D13s317 94.56 0.9983

77891.20 n/a 7870.15 30.52 6300.68 567.26 9447.96 297.27 7872.93 157

9111.40 n/a 1100.10 327.54 1056.42 n/a 1149.91 n/a 1102.15 4

852.42 8.51 107.06 24.56 122.81 14.59 79.29 19.14 103.05 2

D16s539 96.12 0.9995

120297.00 3919.65 14000.16 1223.01 8679.44 392.36 13013.40 249.07 11897.67 283

11841.90 76.10 1871.50 97.13 1219.60 45.35 1643.34 1.02 1578.15 33

1131.67 56.10 172.27 9.78 196.11 7.80 124.13 1.52 164.17 3

Average Targeted D

NFS mtDNA 92.41 0.9969

81232.10 n/a 5730.84 78.66 8416.19 735.35 10454.52 780.82 8200.52 236

7764.42 731.00 1304.40 211.85 1224.56 n/a 1531.58 n/a 1353.51 15

905.77 5.11 187.49 n/a 144.34 2.08 85.86 23.86 139.23 5

Average Non-Targeted D

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Table 3. Retention of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mt

using the Modified Indirect Fishing Protocol #2 (modified hybridization, 2X M-270 beads, modified bead 

Hybridization 1 Hybridization 2 Hybridization 3 Ave

E 

R 

2 Copies In SD 

Copies

Out SD

Copies

Out SD

Copies

Out SD

Copies

Out 

Amelogenin 95.75 0.9994

115459.00 1497.85 17120.90 876.00 23768.20 4069.11 19377.40 1992.22 20088.83 338

10080.00 909.00 2889.33 457.00 1470.43 556.00 1967.21 191.00 2108.99 72

1077.60 4.19 190.12 13.50 292.50 22.50 305.62 13.40 262.74 6

D13s317 95.89 0.9983

78569.90 n/a 16636.10 1785.21 19084.40 1619.12 21092.00 n/a 18937.50 223

8877.80 691.00 1856.82 427.00 1849.81 n/a 1449.64 131.00 1718.76 23

876.08 24.40 228.69 8.80 271.11 51.10 325.75 47.80 275.18 4

D16s539 96.41 0.9993

129002.00 2567.76 13249.20 2868.09 17747.30 669.00 15741.60 4450.15 15579.37 225

11557.90 620.00 2588.96 1321.11 2432.83 551.00 2717.58 850.00 2579.79 14

1232.71 39.70 231.26 49.80 292.16 65.20 339.60 145.00 287.67 5

Average Targeted 

NFS mtDNA 92.68 0.9982

79168.20 9571.68 14529.50 2191.49 17877.50 6353.05 20538.80 4448.40 17648.60 301

8902.93 1319.44 2548.53 1534.00 1526.77 240.00 1820.20 n/a 1965.17 52

941.08 6.35 224.65 14.30 235.59 15.00 235.71 43.20 231.98 

Average Non-Targeted 

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Table 4. Retention of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mt

using the Direct Fishing Protocol 

Hybridization 1 Hybridization 2 Hybridization 3 Average 

E 

R 

2 Copies In SD Copies Out SD Copies Out SD Copies Out SD Copies Out SD 

Amelogenin 94.43 0.9983

106797.00 929.00 20.33 1.84 25.56 0.59 73.39 4.05 39.76 29.24 

10847.50 316.00 2.51 0.09 1.18 0.39 2.11 0.58 1.93 0.68 

954.01 33.90 0.25 0.00 0.09 0.04 0.16 0.02 0.17 0.08 

D13s317 91.33 0.9983

88762.00 5997.40 86.03 3.63 137.90 5.92 246.48 n/a 156.80 81.88 

8814.22 145.00 17.33 n/a 5.24 n/a 10.70 n/a 11.09 6.05 

865.28 24.30 0.67 0.01 1.42 0.94 1.61 0.29 1.23 0.50 

D16s539 95.15 0.9984

124962.00 5160.58 164.32 0.94 253.47 0.15 170.51 19.30 196.10 49.78 

12957.70 59.10 12.19 0.50 8.04 0.51 22.24 0.95 14.16 7.30 

1096.78 57.30 1.33 0.37 0.90 0.42 1.88 0.01 1.37 0.49 

Average Targeted DNA 

NFS mtDNA 90.20 0.9994

86532.50 1260.36 88.64 1.09 131.21 11.40 148.18 2.55 122.68 30.67 

8683.78 109.00 7.73 0.71 7.69 n/a 16.06 2.22 10.49 4.82 

750.00 30.40 0.82 n/a 0.64 0.05 4.09 4.18 1.85 1.94 

Average Non-Targeted DNA 

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Table 5. Loss of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mtDNA

attributable to the Hybridization Only from Indirect Fishing Method

Hybridization 1 Hybridization 2 Average % 

E 

R Loss

% 

2 Copies In SD Copies Out SD Copies Out SD Copies Out SD 

Amelogenin 97.51 0.9992

122537.00 1599.63 10034.12 233.91 6920.24 437.29 8477.18 2201.84 89.38 

11140.70 222.00 1248.69 26.07 706.03 22.15 977.36 383.72 90.34 

1065.88 41.30 90.73 8.33 128.73 8.72 109.73 26.87 90.07 

D13s317 94.14 0.9987

79142.40 n/a 13692.40 1422.70 10753.80 999.06 12223.10 2077.90 75.52 

8685.10 748.00 2091.01 45.23 1208.34 59.67 1649.67 624.14 78.30 

915.41 3.75 127.64 6.29 148.20 26.85 137.92 14.54 77.87 

D16s539 95.02 0.9991

136766.00 3856.13 16992.44 1271.72 15931.84 307.05 16462.14 749.96 84.03 

11638.90 78.60 2334.66 64.39 1207.10 131.75 1770.88 797.30 84.93 

1281.71 5.06 149.02 12.12 184.80 9.07 166.91 25.29 83.31 

NFS mtDNA 90.61 0.9964

102430.00 n/a 11319.88 1474.69 9368.60 3728.48 10344.24 1379.76 74.90 

10111.60 1353.64 1735.15 162.87 944.52 3.69 1339.83 559.06 84.53 

1046.37 126.00 116.66 8.68 146.43 43.44 131.55 21.05 84.56 

Average 83.15 

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Table 6. Loss of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mtDNA

attributable to the Hybridization Only from Modified Indirect Fishing Protocol #2 

Hybridization 1 Hybridization 2 Average 

E 

R

% Loss

% L

2 Copies In SD Copies Out SD Copies Out SD Copies Out SD 

Amelogenin 97.51 0.9992

122537.00 1599.63 10034.12 233.91 6920.24 437.29 8477.18 2201.84 93.08 

11140.70 222.00 1248.69 26.07 706.03 22.15 977.36 383.72 91.23 

1065.88 41.30 90.73 8.33 128.73 8.72 109.73 26.87 89.71 

D13s317 94.14 0.9987

79142.40 n/a 13692.40 1422.70 10753.80 999.06 12223.10 2077.90 84.56 

8685.10 748.00 2091.01 45.23 1208.34 59.67 1649.67 624.14 81.01 

915.41 3.75 127.64 6.29 148.20 26.85 137.92 14.54 84.93 

D16s539 95.02 0.9991

136766.00 3856.13 16992.44 1271.72 15931.84 307.05 16462.14 749.96 87.96 

11638.90 78.60 2334.66 64.39 1207.10 131.75 1770.88 797.30 84.78 

1281.71 5.06 149.02 12.12 184.80 9.07 166.91 25.29 86.98 

NFS mtDNA 90.61 0.9964

102430.00 n/a 11319.88 1474.69 9368.60 3728.48 10344.24 1379.76 89.90 

10111.60 1353.64 1735.15 162.87 944.52 3.69 1339.83 559.06 86.75 

1046.37 126.00 116.66 8.68 146.43 43.44 131.55 21.05 87.43 

Average 87.36 

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Figure 1. Depiction of the Indirect Fishing Protocol (largely following Anderung et 

al (2008). Target molecules are green (in this study, representing the 16 fragments 

targeted by PowerPlex 16® System), non-target molecules are red (in this study, 

representing NFS mtDNA), biotinylated probes are blue, and the streptavidin coated 

magnetic beads are white.

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55
Figure 2. Schematic of methods used to quantify percent retention and loss of DNA standards subject t

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Figure 3. Depiction of the Direct Fishing Protocol. Target molecules are green (in 

this study, representing the 16 fragments targeted by PowerPlex 16® System), nontarget molecules are red (in this study, representing NFS mtDNA), biotinylated probes 

are blue, and the streptavidin coated magnetic beads are white.

57

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Chapter 3: Evaluating the Efficiency of Primer Extension 

Capture as a Method to Enrich DNA Extractions 

Misa Wintersa,b, Cara Monroea, Jodi Lynn Bartac, and Brian M. Kempa,b

a Department of Anthropology, Washington State University, Pullman, WA 99164-4236 

b School of Biological Sciences, Washington State University, Pullman, WA 99164-4910 c Department of Biological and Health Sciences, Madonna University, Livonia, MI 48150

Corresponding Author: 

Brian M. Kemp 

Department of Anthropology 

Washington State University 

Pullman, WA 99164 

Office: 509-335-7403 

Fax: 509-335-3999 

bmkemp@wsu.edu

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58
ABSTRACT

In this study we sought to document the efficiency of primer extension capture (PEC) as 

a method to enrich DNA extractions by two measures, first in its ability to retain targeted 

DNA molecules and, secondly, in its ability to remove non-target DNA molecules from a 

common pool containing both. Efficiencies were estimated following the approach of 

comparing number of “copies in” to “copies out” by quantitative polymerase chain 

reaction (qPCR). Conducted as previously described, PEC retention of DNA targets 

ranging 109-288 base pairs (bps) in length was only 15.88-2.14% (i.e., loss of 84.12-

97.86%). Experimental modifications of PEC aimed to potentially increase this efficiency 

were generally of no avail. However, the benefit of PEC, as originally designed, is in its

ability to remove most non-target DNA molecules (99.99%). We discovered that many 

(56.69%) of the target molecules are “lost” prior to their immobilization on the 

streptavidin-coated beads, that is following extension and purification of the extended 

products. Our results will allow researchers to carefully weigh the general inefficiency of 

PEC in retaining target molecules over its high efficiency in removing non-target 

molecules. Moreover, these efficiencies are directly comparable to previous ones 

estimate for “fishing” for DNA, any alternative method for DNA enrichment. 

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59
INTRODUCTION 

High throughput DNA sequencing [also commonly referred to as next generation 

sequencing (NGS) or massive parallel sequencing (MPS)] holds promise for the 

continued study of low copy number (LCN) and degraded DNA samples. While forensic 

DNA practitioners recognize its utility (e.g., Chang et al., 2009; Butler, 2010; Berglund et 

al., 2011; Parson et al., 2013; Melton, 2014; Yang et al., 2014), it has already been 

established as a reliable approach to the study ancient DNA (aDNA). Reports of the first 

two ancient genomes, a ~4,000 year old Paleo-Eskimo genome (Rasmussen et al.,

2010) and that of the Neanderthal (Green et al., 2010), has led to the rise of a field now 

dubbed “Paleogenomics” (Shapiro and Hofreiter, 2014). Recently Raghavan et al. 

(2014) was able to conduct population level genetic analysis of genomic data, serving as 

an example for the utility of paleogenomics. 

However, with short tandem repeats (STRs) as the current gold standard for forensic 

identification, it has been unclear how soon high throughput sequencing (HTS) would

become widely used by the forensic DNA community, despite its advantages (Holland et 

al., 2009; Butler, 2010; Yang et al., 2014). One obvious drawback of “standard” HTS is 

that it is analogous to screening DNA prior to the invention of PCR, in that, it is not 

directed at molecules of interest, but rather represents a “shot gun” approach. Thus,

standard HTS is not well suited for screening specific markers of interest (i.e., with much 

coverage at any particular site) or mapping repetitive units (Metzker, 2010), such as 

those represented by the STRs in the Combined DNA Index System (CODIS) panel. 

However, forensic researchers have recently demonstrated that, following STR multiplex 

amplification and indexed library preparation of those amplicons, HTS can be leveraged 

to produce reliable STR profiles of adequate depth (Zeng et al., 2015). Here coverage 

(or depth) refers to the number of molecules screened. For example, with HTS, if a site 

has two or more times coverage, the site has been observed on two or more 

independent molecules derived from the biological material from which they have been 

purified. This is vastly different than screening markers with PCR, which theoretically

begins from a pool of molecules intact enough to contain both priming sites. In this 

case, the copy number available [e.g., as assessed by quantitative PCR (qPCR)] from a 

sample dictates the “coverage”, which is observed downstream as an average in the 

amplicons sequenced using the Sanger method or typed by other means (e.g., RFLP, 

length variance). 

As another means to leverage the power of HTS, aDNA researchers have developed a 

variety of “DNA capture” methods that allow for pre-selection of regions of interest, which 

can be later subjected to HTS (e.g., Briggs et al., 2009; Maricic et al., 2010; Carpenter et 

al., 2013). DNA capture is also commonly referred to as “sample enrichment”, “pull 

down”, or “fishing”. Most DNA capture methods, while variable, follow the same general 

principles. First, the DNA sample is denatured and target molecules are hybridized to 

synthetic biotinylated DNA molecules (called “probes” or “bait”). Immobilization of this 

hybridized complex on streptavidin coated magnetic beads, effectively “captures” the

targets. A strong magnet is used to attract the beads to the side or bottom of the tube. 

Buffers are used to wash away impurities and non-target DNA, and then the beads are 

released from the magnet and the target DNA disassociated from the probes. In the 

end, there should be enrichment of target over non-target DNA molecules, and it should 

also be free of impurities (e.g., PCR inhibitors). 

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60
 

While most DNA capture methods have been developed to enrich from aDNA libraries 

(i.e., already amplified DNA) that typically contain comparatively little endogenous DNA 

(Carpenter et al., 2013), it is possible that similar methods could be used to capture 

target molecules from a sample’s total DNA eluate, that is, to prescreen and enrich for

DNA markers of interest prior to PCR amplification or even library builds. Taking this 

perspective it is essential to further consider that when targeting CODIS markers (or any 

set of markers) by traditional PCR, that these reactions contain far more non-target 

genomic DNA of the individual than target DNA [not to mention the possible complicating 

factor of non-target exogenous DNA associated with the sample (Noonan et al., 2005; 

Poinar et al., 2006)]. For example, the sum of the average amplicon lengths targeted in 

Promega Male 9948 DNA by the Promega PowerPlex 16® System is 3573 base pairs 

(bps) (Promega, 2008; Kemp et al., 2014) (Chapter 1). Given that the human genome is 

~3.2 billion bps in length, only ~1.12x10-4% of it need be present in a DNA extract to 

generate a complete profile. In other words, the vast majority of the genome is of no 

consequence to generating a full CODIS profile. In general, amplification efficiency of 

targeted DNA is sub-optimal because non-target DNA can interfere between polymerase 

and targeted DNA molecules and/or compete as binding sites for primers (Wilson, 1997; 

Nielsen et al., 2008). If “capture extracted” DNA is efficient, it could possibly lead to 

more reliable DNA profiling. 

Anderung et al (2008) described one such method of “fishing” for DNA with biotinylated 

probes from a pool of DNA extracted from ancient cattle bones and teeth. Nandineni 

and Vedanayagam (2009) also used a fishing approach to capture CODIS STRs. In 

both studies, the researchers demonstrated the feasibility of the methods, but did not 

document its efficiency against a standard (e.g., Barta et al., 2014; Kemp et al., 2014) 

(Chapter 1). Using such an approach, Winters et al. (In Prep) (Chapter 2) recently 

observed that the method is generally inefficient at both retaining target molecules and 

removing non-target molecules. Through a series of experiments, the best average 

retention of DNA targets ranging 109-288 bps in length was 31.41-12.08%, which was 

similar to the average retention of non-target molecules (22.07-24.65%, two tailed t-test 

p=0.623). While the method may be useful in the study of DNA compromised by 

polymerase chain reaction (PCR) inhibitors (Wang and McCord, 2011), one must 

carefully weigh this possible advantage against the degree of expected DNA loss and 

the non-selectivity of the method for targeted over non-targeted DNA.

Primer extension capture (PEC) is an alternative capture method developed by Briggs et 

al (2009) (Figure 1). The PEC method expands on fishing for DNA with the addition of 

one cycle of annealing and polymerase extension of the biotinylated probes on the target 

molecules. Following extension, the biotinylated probes become complementary to the 

length of the target molecule. Thus, the target DNA should be more tightly bound to the 

probes, in comparison to fishing for DNA with short probes. After primer extension the 

products are immobilized onto streptavidin coated magnetic beads and separated from 

the non-target DNA and impurities present in the supernatant. Briggs et al. (2009) used 

this method to capture and sequence pieces of the complete Neanderthal mitochondrial 

genome. 

In this study we sought to document, by two measures, the efficiency of PEC as an 

extraction method, first in its ability to retain targeted DNA molecules and secondly in its 

ability to remove non-target DNA molecules. Efficiencies were estimated following the 

approach of comparing number of “copies in” to “copies out” by qPCR (Barta et al., 

2014; Kemp et al., 2014) (CHAPTER 1). Since the efficiency of fishing for DNA as a 

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61
 

generalized pre-PCR extraction method has already been documented in this manner, it 

permits direct evaluation of PEC against that method (Winters et al., In Prep) (Chapter 

2). 

MATERIALS AND METHODS

Creation of the Pooled Standards 

Pooled standards containing the 16 fragments of genomic DNA amplified by the 

Promega PowerPlex 16® System (i.e., representing targeted DNA for PEC) and a 181 bp 

fragment of northern fur seal mitochondrial DNA (i.e., representing non-targeted DNA, 

and abbreviated “NSF mtDNA” throughout the remainder of the study) were created 

following Winters et al (In Prep) (Chapter 2) to final concentrations of 1 ×	105, 1 ×	104, 

and 1 ×	103 copies/μL. For example, the 1x103 pooled standard contained 1000 copies 

of each of the sixteen PowerPlex 16® System amplicons and 1000 copies of NFS mtDNA 

per microliter. 

Primer Extension Capture [Largely Following Briggs et al. (2009)] (Figure 1) 

Primer Extension 

One hundred microliter PCRs contained: 1X Gold Buffer, 2.5 mM MgCl2, 250 M of each 

dNTP, 200 g/L BSA, 0.05 pmol of each of the 32 Promega PowerPlex 16® System 

biotinylated primers (Winters et al., In Prep) (Chapter 2), 5 U Amplitaq® Gold, and 30 μL 

of pooled standard DNA. One round of PCR was conducted (i.e., to extend the 

biotinylated primers). Following twelve minutes of activation at 95°C, reactions were 

held at 60°C for one minute, followed by a two minute hold at 72°C. 

Primer extension products were purified with the Qiagen MinElute® PCR Purification Kit, 

following the manufacturer’s instructions except that the pH indicator was not added.

Final elution was performed in 30 μL of Buffer EB. 

Bead Preparation 

Two hundred and fifty microliters of Dynabeads® M-270 Streptavidin-coated beads were 

prepared by placing them on the magnet (DynamagTM) for 2 minutes and removing the 

supernatant. The beads were then washed in 500 μL of 2X Binding and Wash (B&W) 

buffer [10mM Tris-HCl pH 8.0, 1mM EDTA, 1M NaCl, 0.2% Tween-20 (v/v) — it is 

notable that the recipe of Briggs et al. (2009) for B&W buffer differs from that described

by Invitrogen (2012)]. The mixture was vortexed and placed back on the magnet for 2 

minutes and the supernatant removed. This wash was repeated and, subsequently, the 

beads were resuspended in 250 μL of 2X B&W buffer. 

Immobilization/Capture 

A 25 μL aliquot of the prepared beads was added to the purified primer extension 

products. This mixture was allowed to rotate at room temperature for 30 minutes. The 

immobilized mixture was transferred to a new 1.5 mL tube to which 500 μL of 1X B&W 

buffer was added. This was placed on the magnet for 2 minutes and the supernatant 

removed. Washing with 1X B&W buffer in this manner was repeated an additional four 

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62
times. Following the fifth wash, 500 μL of “hot wash” buffer [1X Amplitaq Gold Buffer, 

2.5 mM MgCl2, 0.1% Tween-20 (v/v)] were mixed with the beads and this volume 

transferred to a new 1.5 mL tube. The tube was rotated at 65°C for two minutes, placed 

on the magnet for two minutes, and the supernatant removed. Beads were resuspended 

in 30 μL of Buffer EB and the mixture heated for 2 minutes and 30 seconds at 95°C. 

The tube was immediately placed back on the magnet and the supernatant was 

collected and placed in a new 1.5 mL tube after one minute. The eluate volume was 

brought to 50 μL with dH2O. 

Modified Primer Extension Capture #1 (Platinum® Taq, Modified Wash Following 

Capture) 

In this modified version of the Briggs et al. (2009) PEC, we substituted Platinum® Taq in 

the primer extension step and used a modified final wash step prior to elution. 

Primer Extension 

One hundred microliter PCRs contained: 1X PCR Buffer, 1.5 mM MgCl2, 0.32 M of 

each dNTP, 0.05 pmol of each of the 32 Promega PowerPlex 16® System biotinylated 

primers (Winters et al., In Prep) (Chapter 2), 1.9 U Platinum® Taq, and 60 μL of pooled 

standard DNA. Following a three minute hold at 94°C, reactions were held at 94°C for 

15 seconds, 60°C for 1 minute, and 72°C for 2 minutes. After returning to room 

temperature, volumes were transferred to 1.5 mL tubes. Primer extension products 

were purified as described above for the primer extension capture protocol. 

Bead Preparation 

Beads were prepared as described for the primer extension capture protocol. 

Immobilization/Capture 

Capture and final elution were conducted as described for the primer extension capture 

protocol, except that following the fifth wash, the beads were washed with 500 μL of 1X

Invitrogen PCR buffer. 

Modified Primer Extension Capture #2 (Platinum® Taq, Primer Extension Products 

Not Purified, Modified Beads and Capture) 

In this modified version of the Briggs et al. (2009) PEC, we substituted Platinum® Taq in 

the primer extension step. The primer extension products were not purified prior to 

capture and we modified the final wash and elution steps.

Primer Extension 

Composition of PCRs followed that described for modified PEC #1. Following a three 

minute hold at 94°C, reactions were held at 94°C for 15 seconds, 60°C for 15 seconds, 

and 72°C for 15 seconds. After returning to room temperature, volumes were 

transferred to 1.5 mL tubes. 

Bead Preparation 

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63
Eighty microliters of Dynabeads® M-270 Streptavidin-coated beads were prepared by 

adding 200 μL of 1X Binding and Wash (B&W) buffer [following Invitrogen (2012): 5 mM 

Tris-HCl pH 7.5, 0.5 mM EDTA, 1M NaCl], vortexing the tube, placing it on the magnet 

for 2 minutes, and removing the supernatant. This was repeated two additional times for 

a total of three washes with 1X B&W buffer. Finally, beads were eluted in 1000 μL of 2X

B&W buffer. 

Immobilization/Capture 

A 100 μL aliquot of the prepared beads was added to the 100 μL of primer extension 

products. This results in a 1X final concentration of B&W buffer, the ideal condition (i.e., 

1 M NaCl) for immobilization (Invitrogen, 2012). This mixture was rocked gently at room 

temperature for 30 minutes before being placed back on the magnet for 2 minutes, 

which was followed by three washes with 200 μL of 1X B&W. The beads were eluted in 

60 μL with dH2O. This volume was heated to 95°C for five minutes, immediately placed 

on the magnet, and, after one minute, the supernatant was collected into a new 1.5 mL 

tube. 

Modified Primer Extension Capture #3 (Platinum® Taq, Primer Extension Products 

Not Purified, 2nd Modified Wash and Elution) 

In this modified version of the Briggs et al. (2009) PEC, we substituted Platinum® Taq 

for the primer extension. The primer extension products were not purified prior to 

immobilization and we modified the final wash and elution steps over that described for 

the modified PEC #2. 

Primer Extension 

Composition of PCRs and conditions were as described for the modified PEC #1. 

Bead Preparation 

Beads were as described for the modified PEC #2. 

Immobilization/Capture 

Immobilization was modified over that described for the modified PEC #2. Following the 

three washes with 200 μL of 1X B&W, the beads were resuspended in 500 μL of hot 

wash buffer and the volume transferred to a new 1.5 mL tube. The tube was rotated at

65°C for two minutes, placed on the magnet for two minutes, and the supernatant 

removed. Beads were resuspended in 60 μL of dH2O and the mixture heated for five 

minutes at 95°C. The tube was immediately placed back on the magnet and the 60 μL 

of supernatant was collected and placed in a new 1.5 mL tube after one minute. 

Analysis of Primer Extension Efficiency Between Amplitaq® Gold and Platinum® 

Taq 

Thirty microliters of pooled standard were subjected to primer extension with Amplitaq® 

Gold or Platinum® Taq and purified with the Qiagen MinElute® PCR Purification Kit as

described above for primer extension capture. Final elutions were performed with 30 μL 

Buffer EB, then eluate volumes were brought to 50 μL with dH2O. 

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64
 

Quantitative PCR (qPCR) and Calculating Percent Retention and Loss 

The amount of Amelogenin, D13s317, D16s539, and NFS DNA remaining in the 

standard after experimental treatment (i.e., “copies out”) was determined by qPCR 

following Winters et al (Winters et al., In Prep) (Chapter 2). Quantification of each 

marker was conducted in duplicate against standard curves generated from two 

reactions each of standard pools at 1 ×	105, 1 ×	104, 5 ×	103, 2 ×	103, 1 ×	103, 5 ×	102, 

2.5 ×	102, 1 ×	102 and 50 copies/μL. Two additional 1 ×	105, 1 ×	104, and 1 ×	103

copies/μL standards (untreated) each were quantified as unknowns to monitor the 

concentration of pre-experimental standards. These provide estimates of “copies in”. 

Subtraction of the number of “copies out” (measured as the average of duplicate qPCR 

amplifications from the molecules retained following purification) from “copies in” 

(measured as the average of duplicate qPCR amplifications of the standards treated as 

unknowns) divided by “copies in” multiplied by 100 provides the percent retention:

[(copies in - copies out)/ copies in] ×	100 

One hundred minus retention provides a measure of percent loss. Retention and loss of 

Amelogenin, D13s317, D16s539, and NFS mtDNA was determined in this manner three 

times, from which the average loss and the uncorrected sample standard deviation (i.e., 

the standard deviation of the sample) were calculated. 

Statistical Analyses 

Two-tailed t-tests were used to statistically evaluate differences in mean percentage 

retention at the 0.05 level of probability. Statistical analyses were conducted in StatPlus 

(AnalystSoft, Inc.).

RESULTS 

Quantitative PCR efficiencies for Amelogenin, D13s317, and D16s539 ranged from 

92.64-98.08%, with R2 values ranging 0.9988-0.9994 (Tables 1-6), comparable to 

previous observations (Kemp et al., 2014; Winters et al., In Prep) (Chapters 1 & 2). The 

NSF mtDNA qPCR efficiencies ranged from 96.62-93.06% with R2 values from 0.9965-

0.9991, also comparable to previous observations Winters et al (In Prep) (Chapter 2). 

Across all three standards (1 ×	105, 1 ×	104, and 1 ×	103 copies/μL), average DNA 

retention of targeted Amelogenin, D13s317, and D16s539 DNA following PEC was 

7.66% (i.e., loss of 92.34%) (Table 1). Almost all of the non-targeted NFS mtDNA was 

removed during the process, with an average loss of 99.99%, which is statistically more 

loss than that of the targeted molecules (p=0.037). Subjected to modified PEC #1, 

1.72% of the targets were retained (i.e., loss of 98.28%) and 99.98% of non-target 

molecules removed (Table 2). Average loss of target and non-target molecules was not 

statistically different (p=0.075) following modified PEC #1. Subjected to modified PEC 

#2, 10.90% of the targets were retained (i.e., loss of 89.10%) and 90.65% of non-target 

molecules removed (Table 3). Average loss of target and non-target molecules was not 

statistically different (p=0.698) following modified PEC #2. Modified PEC #3 was 

associated with the retention of 7.47% of the targets (i.e., loss of 92.53%) and a loss of 

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65
99.59% of non-target molecules (Table 4). Average loss of target and non-target 

molecules was not statistically different (p=0.023). 

The mean retention of targeted molecules by PEC (7.66%) was larger than that of 

modified PEC # 1 (1.72%, p=0.005), but not statistically different from that retained by

modified PEC #2 (10.90%, p=0.265) or from modified PEC #3 (7.47%, p=0.936). The 

mean loss of non-target molecules by PEC (99.99%) was not different from that lost 

following modified PEC # 1 (99.98%, p=0.932), but statistically greater than the average 

loss associated with modified PEC #2 (90.65%, p=2.5 ×	10-6) and modified PEC #3 

(99.59%, p=0.003). 

On average, more than half of the target DNA in the standards (56.59%) subjected 

solely to primer extension with Amplitaq® Gold, followed by purification with the Qiagen 

MinElute® PCR Purification Kit, were lost (Table 5). This was statically indistinguishable 

from the loss of the non-target DNA (65.78%, p=0.237). Less than half of the target 

DNA in the standards was lost (45.26%, but not statistically different from Amplitaq® 

Gold results, p=0.054) when subject to primer extension with Platinum® Taq, followed

by purification with the Qiagen MinElute® PCR Purification Kit. Loss of non-target DNA 

(50.18%) was not statistically different that loss of target DNA (p=0.628). 

DISCUSSION 

In this study we sought to document the efficiency of PEC (Figure 1) as an extraction 

enrichment method by two measures, first in its ability to retain targeted DNA molecules 

and, secondly, in its ability to remove non-target DNA molecules. Following the protocol 

of Briggs et al (2009) results in the loss of 92.34% of the three targeted molecules 

(Amelogenin, D13s317, and D16s539). Since these three markers were chosen as 

representatives of the fragments targeted for amplification by the Promega PowerPlex 

16® System, presumably the same percentage of the other thirteen targets was lost. 

While PEC might be considered largely inefficient, the method removes proportionally 

more non-target DNA molecules (i.e., 99.99% of NFS mtDNA molecules were removed, 

p=0.037). This is precisely why PEC can be of value, in focusing the power of HTS on 

molecules of interest over non-target regions of the genome and also act to exclude 

DNA exogenous to the individual under study. Regardless of absolute efficiency, PEC is 

probably more efficient at removing non-target DNA because, even if the probes were 

able to anneal to non-target molecules, mismatches at or near the 3’ end of the primers 

would result in poor extension of the molecule by the polymerase (Palumbi, 1996). 

Thus, they would be subsequently more difficult to immobilize. 

Across our experimental modifications to the PEC protocol, none were equivalent to the 

original protocol. Modified PEC #1 retained less target molecules (1.72%, p=0.005) but 

was equivalent in its removal of non-target molecules (99.98%, p=0.932). Both modified 

PEC #2 and #3 were equivalent in their retention of CODIS molecules (10.90%, p=0.265 

and 7.47%, p=0.936, respectively), but associated with less efficient removal of the NSF 

mtDNA molecules (90.65%, p=2.5 ×	10-6 and 99.59, p=0.003, respectively). Thus, 

based on the results of our study, if PEC is to be used as a DNA capture method, it is 

recommendable to follow the protocol as originally described (Briggs et al., 2009). 

However, it is notable that employment of Amplitaq® Gold may not be ideal with all 

samples and/or sample types, as previous investigation has shown that this polymerase 

is particularly prone to inhibition (Monroe et al., 2013). 

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66
Further modification to the basic PEC method should be directed at improving the 

retention of target molecules, as was possible with modifications to the fishing method 

(Winters et al., In Prep) (Chapter 2). However, while the optimized fishing protocol 

retained on average more target molecules (21.70%, p=4 ×	10-5) than possible with 

PEC, removal of non-target molecules reach only 76.99% (thus, it inadvertently retained 

far more non-target DNA, 23.01%, p=9.8 ×	10-6). This illustrates a strength in our 

assessment of efficiency using common standards; the outcomes are directly 

comparable, resulting in both relative and absolute assessments of efficiency. 

Our unique approach to quantifying efficiency by comparing “copies in” to “copies out”

estimated from quantitative PCR (Barta et al., 2014; Kemp et al., 2014) (Chapter 1) 

permitted us to begin identifying wherein DNA loss occurs during the PEC protocol. 

Specifically, we observed an average loss of 56.59% of the target DNA following 

extension by Amplitaq® Gold and purification with the Qiagen MinElute® PCR 

Purification Kit (Table 5). Previously, losses of Amelogenin, D13s317, and D16s539 at 

52%, 40.85%, and 38.95% (average loss across pooled standards starting at 1x107, 

1x105, and 1x104 copies/μL) have been observed following purification with the Qiagen 

MinElute® PCR Purification Kit (Kemp et al., 2014) (Chapter 1). From this, it might be 

concluded that the remaining fraction of lost molecules (~4-17%) is attributable to the 

twelve minute activation at 95°C required by the enzyme. More appropriately, these lost 

molecules might be better termed “unamplifiable”, probably having lost one or both 

priming sites as a result of degradation of the single strands during heat activation. Yet, 

these numbers are not additive, given the theoretical expectation that primer extension 

should double the number of target molecules. Working through an example will aid in 

illuminating this point.

The average copy number of D16s539 retained from the 1 ×	105 standard was 67124.71 

per microliter. If 38.95% loss is attributable to purification by the Qiagen MinElute® PCR 

Purification Kit, there should have been 172335.58 copies present prior to purification, 

representing only a 42.9% rise over the pre-amplification count of 120591 copies. 

Moreover, since NFS mtDNA copies should not have increased following one round of 

extension (i.e., because northern fur seal probes were not added), it is hard to reconcile 

why loss of these molecules (58.89%) was not greater than that observed for target 

molecules (p=0.237). Perhaps all of these numbers are not simply additive because of 

the loss of amplifiable molecules due to the prolonged heat activation step necessary for 

the polymerase that leaves the DNA exposed while in a state of single strandedness. 

Therefore, it is reasonable to conclude that primer extension probably does not double 

the copy number, which uniquely contributes to the overall low efficiency of PEC in 

retaining molecules of interest compared to fishing for DNA. Since losses associated

with extension by Platinum® Taq were not statistically different from that employing 

Amplitaq® Gold, this substitution provides no relief from the heat activation issue.

It is worth emphasizing that the outcome of the experiments described here were not 

compared with standard methods of DNA extraction and genotyping, which would have 

pointed to the strength and weakness of PEC over standard methods. Nevertheless, our 

study is the first to evaluate the efficiency of PEC for DNA against 100% recovery of 

molecules sized exactly for amplification with the Promega PowerPlex 16® System. A 

possible future direction would be to experimentally degrade native DNA (e.g., Ambers 

et al., 2014) and evaluate the efficiency of PEC for capture of target molecules from total 

genomic DNA that is degraded and chemically modified.

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

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necessarily reflect the official position or policies of the U.S. Department of Justice.

67
While PEC is an established method for enriching target DNA from libraries prior to HTS

(Briggs et al., 2009), our experiments raise concern over its use as method for enriching 

DNA extractions prior to PCR amplification. Before adopting this method, researchers 

should carefully weigh its general inefficiency in retaining target molecules over its high 

efficiency in removing non-target molecules.

ACKNOWLEDGMENTS 

Thanks to Kathleen Judd for assistance in the laboratory. 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

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68
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70
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71
Table 1. Retention of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mt

using primer extension capture.

Capture 1 Capture 2 Capture 3 Ave

E

R2 Copies In SD 

Copies

Out SD Copies Out SD 

Copies

Out SD

Copies

Out 

Amelogenin 97.53 0.9993

111394.00 2364.17 2964.54 58.87 2273.65 n/a 2170.82 239.57 2469.67 43

11340.70 481.00 235.92 8.95 217.99 52.94 272.59 9.39 242.17 2

1072.08 47.40 21.99 2.31 27.99 0.19 23.23 3.22 24.40 

D13s317 93.24 0.9993

82378.40 11532.43 1370.28 11071.29 50.81 9593.43 1298.55 10732.39 101

8294.56 943.00 1008.36 179.15 1237.77 1181.44 111.11 1142.52 11

841.48 52.50 130.71 10.80 140.45 6.13 129.65 5.75 133.60 

D16s539 96.26 0.9991

115868.00 4075.82 7427.77 248.48 6422.53 435.67 6310.06 212.29 6720.12 61

11735.90 358.00 658.02 6.03 768.50 115.41 865.75 135.18 764.09 10

1099.80 2.63 68.92 0.67 84.98 11.50 87.32 0.59 80.40 1

Average Targeted 

NFS mtDNA 90.78 0.9980

70310.00 n/a 1.15 0.08 1.25 1.14 1.21 0.80 1.20 

8222.18 1490.78 0.32 0.19 0.71 0.41 0.14 0.17 0.39 

905.29 73.50 0.09 n/a undetermined n/a 0.22 0.02 0.15 

Average Non-Targeted 

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Table 2. Retention of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mt

using the modified primer extension capture #1 (Platinum® Taq, modified wash following capture)

Capture 1 Capture 2 Capture 3 Averag

E 

R 

2 Copies In SD Copies Out SD Copies Out SD Copies Out SD Copies Out S

Amelogenin 96.54 0.9994

115291.00 704.00 304.77 38.10 481.53 15.00 415.77 12.10 400.69 89.3

9145.61 296.00 29.90 1.73 29.33 3.44 23.75 1.22 27.66 3.3

832.37 43.20 2.65 0.91 2.83 0.72 2.18 0.19 2.55 0.3

D13s317 92.78 0.9993

96424.40 1069.58 2808.71 63.90 4456.11 98.90 3513.25 166.00 3592.69 826.5

7643.45 430.00 283.24 12.40 316.82 11.00 208.75 48.10 269.60 55.3

753.47 43.70 35.40 0.67 21.11 1.67 19.50 0.45 25.33 8.7

D16s539 94.48 0.9990

114253.00 8780.81 1393.46 69.10 1788.37 17.90 1632.64 25.80 1604.82 198.9

8674.43 56.60 121.15 5.09 129.06 0.78 116.72 24.10 122.31 6.2

891.48 74.50 12.40 0.20 8.31 0.29 8.75 1.01 9.82 2.2

Average Targeted DN

NFS mtDNA 86.62 0.9989

86073.40 2627.17 6.19 n/a 9.59 0.90 10.38 2.50 8.72 2.2

7142.19 417.00 0.42 0.29 0.39 0.08 0.34 0.12 0.38 0.0

736.50 15.00 0.40 n/a 0.39 n/a 0.27 0.01 0.35 0.0

Average Non-Targeted DN

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Table 3. Retention of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mt

using the modified primer extension capture #2 (Platinum® Taq, primer extension products not purifiedand capture) 

Capture 1 Capture 2 Capture 3 Aver

E 

R 

2 Copies In SD 

Copies

Out SD

Copies

Out SD

Copies

Out SD

Copies

Out 

Amelogenin 96.69 0.9994

114610.00 47.40 3143.43 111.00 1767.23 26.00 2780.16 2.58 2563.61 71

11290.50 448.00 292.97 12.30 281.91 9.15 349.40 20.80 308.09 3

983.09 80.10 26.44 1.80 32.44 3.85 30.76 2.74 29.88 

D13s317 93.23 0.9991

77567.50 15833.90 812.00 12419.80 258.00 13216.50 3693.68 13823.40 178

8589.38 1040.81 1303.18 312.00 1083.57 395.00 1506.55 274.00 1297.77 21

887.22 53.80 160.48 11.50 144.54 24.70 176.87 0.73 160.63 1

D16s539 96.25 0.9992

128385.00 2112.88 15539.90 235.00 12201.20 186.00 15051.70 585.00 14264.27 180

11548.70 546.00 1439.92 13.00 1483.18 114.00 1691.36 35.50 1538.15 13

1092.93 26.10 147.37 1.54 160.30 0.25 172.88 16.00 160.18 1

Average Targeted D

NFS mtDNA 93.06 0.9991

79960.20 n/a 9473.66 13.80 6732.82 68.40 7354.30 2173.29 7853.59 143

9377.67 647.00 894.68 n/a 753.99 n/a 911.09 n/a 853.25 8

882.16 18.20 80.65 4.23 74.74 n/a 86.03 11.30 80.47 

Average Non-Targeted D

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Table 4. Retention of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mt

using the modified primer extension capture #3 (Platinum® Taq, primer extension products not purified

and elution)

Capture 1 Capture 2 Capture 3 Aver

E 

R 

2 Copies In SD 

Copies

Out SD

Copies

Out SD

Copies

Out SD

Copies

Out 

Amelogenin 96.54 0.9991

99253.60 10778.90 2277.71 18.80 2093.12 183.00 2633.96 362.00 2334.93 27

10814.70 207.00 183.91 n/a 89.95 15.50 217.03 38.30 163.63 6

1023.70 30.80 17.86 1.53 14.31 0.92 10.34 1.44 14.17 

D13s317 93.64 0.9992

91774.60 11837.60 11485.40 736.00 10439.10 445.00 11870.20 1034.46 11264.90 74

9683.25 69.00 1045.31 45.70 1076.99 67.10 1235.51 33.70 1119.27 10

874.93 60.60 108.98 2.27 94.28 0.59 77.01 0.67 93.42 1

D16s539 92.64 0.9994

114193.00 15981.50 10383.30 362.00 9563.37 140.00 11299.90 118.00 10415.52 86

10293.50 352.00 864.72 114.00 805.66 30.90 1201.67 14.70 957.35 21

1028.35 69.20 115.59 1.59 73.05 0.74 90.46 4.96 93.03 2

Average Targeted D

NFS mtDNA 90.96 0.9965

97258.50 1994.34 602.79 184.90 253.37 63.62 243.30 155.87 366.49 20

9489.92 319.00 46.22 9.47 26.10 12.44 17.37 1.56 29.90 1

898.32 8.80 5.74 1.13 4.00 1.67 4.38 1.44 4.70 

Average Non-Targeted D

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

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Table 5. Loss of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mtDNA

following primer extension with Amplitaq Gold ® and Minelute

® purification 

Round 1 Round 2 Average % 

E 

R Loss 2 Copies In SD Copies Out SD Copies Out SD Copies Out SD 

Amelogenin 96.01 0.9994

114718.00 5144.78 35232.75 2100.93 40658.83 n/a 37945.79 3836.82 66.92

11004.50 333.00 3182.93 305.35 2735.33 n/a 2959.13 316.50 73.11

1035.97 3.88 286.37 48.62 346.41 n/a 316.39 42.45 69.46

D13s317 93.36 0.9988

63422.90 n/a 43762.33 8355.65 33143.33 n/a 38452.83 7508.77 39.37

9423.94 n/a 4972.89 1519.04 4094.92 42.69 4533.90 620.82 51.89

952.52 14.20 370.24 39.34 345.01 n/a 357.62 17.83 62.46

D16s539 93.94 0.9989

120591.00 936.00 64514.83 578.65 69734.58 344.24 67124.71 3690.92 44.34

12238.80 454.00 5692.23 49.57 5995.17 387.54 5843.70 214.21 52.25

1215.81 47.20 634.35 10.89 592.43 99.59 613.39 29.64 49.55

Average Targeted DNA 56.59

NFS mtDNA 91.20 0.9986

81128.30 n/a 31073.92 5699.87 35056.42 9125.80 33065.17 2816.05 59.24

9426.45 35.50 3069.90 245.70 3344.06 81.09 3206.98 193.86 65.98

1012.44 35.50 286.49 18.51 278.16 n/a 282.33 5.89 72.11

Average Non-Targeted DNA 65.78

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

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necessarily reflect the official position or policies of the U.S. Department of Justice.
Table 6. Loss of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mtDNA

following primer extension with Platinum Taq

® and Minelute

® purification 

Round 1 Round 2 Round 3 

E 

R 

2 Copies In SD Copies Out SD Copies Out SD Copies Out SD Copies Out 

Amelogenin 98.08 0.9991

126717.00 543.00 52663.50 1867.68 58274.00 681.00 70503.90 2168.71 60480.47 

11855.30 543.00 4622.48 232.00 5381.83 197.00 8257.73 44.10 6087.35 

1091.40 6.38 394.34 0.33 358.22 5.74 470.23 54.50 407.60 

D13s317 92.73 0.9990

72830.60 n/a 49816.20 n/a 52510.20 n/a 58979.30 5866.78 53768.57 

9175.94 n/a 5028.88 231.00 5321.58 489.00 6533.42 1097.68 5627.96 

879.23 25.80 386.82 18.40 382.00 13.00 533.03 433.95 

D16s539 95.84 0.9992

129488.00 2467.53 69658.00 2581.48 74550.20 3908.34 88620.80 3040.57 77609.67 

12265.70 136.00 6008.81 75.50 7830.31 427.00 10506.20 171.00 8115.11 

1130.15 41.60 532.45 23.50 490.53 13.30 523.66 21.10 515.55 

Average Targe

NFS mtDNA 92.36 0.9980

76093.70 n/a 50820.40 n/a 56341.80 n/a 63146.60 5153.41 56769.60 

10229.30 n/a 4714.22 114.00 4775.21 88.20 5258.20 n/a 4915.88 

1000.43 21.40 267.03 4.46 227.25 n/a 309.78 n/a 268.02 

Average Non-Targe

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Figure 1. Depiction of Primer Extension Capture [largely following Briggs et al. 

(2009)] Target molecules are green (in this study, representing the 16 fragments 

targeted by PowerPlex 16® System), non-target molecules are red (in this study, 

representing NFS mtDNA), biotinylated probes are blue, and the streptavidin coated 

magnetic beads are white. 

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78
 

Chapter 4: Evaluating the Efficiency of “Mega-Probe” Capture as

a Method to Enrich DNA Extractions 

Misa Wintersa,b, Cara Monroea, Jodi Lynn Bartac, and Brian M. Kempa,b

a Department of Anthropology, Washington State University, Pullman, WA 99164-4236 

b School of Biological Sciences, Washington State University, Pullman, WA 99164-4910 c Department of Biological and Health Sciences, Madonna University, Livonia, MI 48150

Corresponding Author: 

Brian M. Kemp 

Department of Anthropology 

Washington State University 

Pullman, WA 99164 

Office: 509-335-7403 

Fax: 509-335-3999 

bmkemp@wsu.edu

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79
ABSTRACT

In this study we sought to document the efficiency of a previously described DNA 

capture method, one that we call “mega-probe” capture, for enriching DNA extracts of 

target molecules over non-target ones. Efficiencies were estimated following the 

approach of comparing number of “copies in” to “copies out” by quantitative polymerase 

chain reaction (qPCR). Mega-probe capture increased target molecules from standards 

by 702.46%, an impossible outcome. From our observations of negative controls, 

6217.43, 14555.55, and 36621.00 copies/μL of D16s539, D13s317, and amelogenin bait 

molecules, respectively, became counted as captured copies when, in fact, there were 

no copies to capture. Due to unexpected experimental outcomes, we were not able to 

estimate the efficiency of this method in its removal of non-target molecules. 

Nevertheless, our principal concern about mega-probe capture is the possibility that the 

mega-probe bait becomes counted as captured target molecules. Thus, any attempt to 

measure its efficiency in retaining targeted molecules will be biased. Additional 

experiments conducted on DNA retention following hybridization steps of the megaprobe protocol reveal tremendous loss of molecules attributable to both purification by 

the Qiagen MinElute® PCR Purification Kit and heat exposure. These experiments 

highlight the critical need for those working with LCN and degraded DNA samples to be 

mindful of the loss of already limited DNA strands available for analysis due to the 

experimental treatments used to study them.

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

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80
INTRODUCTION 

Attempts to more fully leverage the power of high throughput sequencing (HTS) of DNA 

[also commonly referred to as next generation sequencing (NGS)] have produced 

various DNA enrichment methods (Briggs et al., 2009; Carpenter et al., 2013). While the 

protocols vary in their details, the end goal of these methods is to enrich target DNA 

molecules (i.e., genomic regions of interest of the organism under investigation) over 

non-target ones (i.e., genomic regions not of interest from the organism under 

investigation, as well as any exogenous DNA present in the extract) from a pool 

containing both. 

Most DNA capture methods follow the same general principles. The DNA sample is 

denatured and target molecules are hybridized to synthetic biotinylated DNA molecules 

(called “probes” or “bait”). The hybridized complex is immobilized on streptavidin coated 

magnetic beads, and a strong magnet is used to attract the beads to the side or bottom 

of the tube. Buffers are used to wash away impurities (e.g., PCR inhibitors) and nontarget DNA, and then the beads are released from the magnet and the target DNA 

disassociated from the probes. The enriched DNA can now be used for downstream 

applications. While many of the capture methods were designed to enrich from DNA 

libraries (i.e., already amplified DNA) prior to HTS (Briggs et al., 2009; Carpenter et al., 

2013), they might also be useful in enriching DNA extracts prior to amplification. In fact, 

the earliest, and arguably the simplest method of capture, known as “fishing” for DNA 

was presented as an alternative extraction method to a far more common silica-based 

method (Anderung et al., 2008). However, prior to adopting any capture method, it 

would be useful to have some estimates of its efficiency.

Winters et al (In Prep-a) (Chapter 2) used a quantitative PCR (qPCR) approach to 

measure the efficiency of “fishing” for DNA targets ranging 109-288 base pairs (bps) in 

length from standards containing known numbers of copies of these target molecules. 

This permitted the efficiency to be measured against 100%, rather than relative against 

some set of alternative methods (Barta et al., 2014; Kemp et al., 2014) (Chapter 1). 

They found that average retention of target molecules reached only 31.41-12.08%, 

which was similar to the average retention of non-target molecules (22.07-24.65%, two 

tailed t-test p=0.623). In other words, fishing for DNA simply lowered the concentration 

of DNA in the extract. Thus, the method would appear to be a poor choice, despite its

potential usefulness for enriching DNA in the presence of inhibitors (Anderung et al., 

2008; Nandineni and Vedanayagam, 2009; Wang and McCord, 2011). Yet, it is 

important to highlight that the absolute efficiencies of many routinely used DNA 

extraction methods are unknown (Barta et al., 2014; Kemp et al., 2014) (Chapter 1). 

Primer extension capture (PEC) is an alternative capture method that builds on the idea 

of “fishing” for DNA with the addition of one cycle of annealing of the biotinylated probes 

and extension with polymerase on targeted molecules (Briggs et al., 2009). 

Theoretically, this should result in target DNA that is bound more tightly to the probes 

over that achieved with fishing with short probes alone. Using a directly comparable 

qPCR approach, Winters et al (In Prep-b) (Chapter 3) determined that PEC retains only 

15.88-2.14% of DNA targets ranging 109-288 base pairs (bps) but simultaneously 

removes 99.99% of non-target molecules. These results permit researchers to carefully 

weigh the general inefficiency of PEC in retaining target molecules against its high 

efficiency in removing non-target molecules.

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81
The capture method of Maricic et al (2010) builds long DNA fragments into biotinylated 

probes that are subsequently used to capture other DNA molecules, those 

complementary to the long probes. We refer to this approach as “mega-probe” capture 

(Figure 1) because it employs biotinylated probes much longer than those used in the 

other two methods described above. Maricic et al (2010) made copies of the full 

mitochondrial genome with two long range polymerase chain reactions (PCRs). The 

amplicons were then sonicated to fragments ~150-850 bps in length. The various sized 

fragments were then purified, biotinylated, and immobilized on streptavidin beads. They 

used their mtDNA mega-probes to capture and sequence 46 complete human mtDNA 

genomes and the method has also been used to capture and sequence the complete 

mitochondrial genome from an extinct Denisovian hominin (Reich et al., 2010). 

Mega-probe capture theoretically holds promise for enrichment of DNA recovered from 

low copy number (LCN) and degraded DNA samples, however it would be instructive to 

estimate its efficiency so as to be directly comparable to efficiency estimates of fishing 

(Winters et al., In Prep-a)(Chapter 2) and PEC (Winters et al., In Prep-b) (Chapter 3). 

Thus, in this study we sought to document, by two measures, the efficiency of megaprobe capture as a method for enriching DNA extractions, first in its ability to retain 

targeted DNA molecules and secondly in its ability to remove non-target DNA molecules. 

Efficiencies were estimated following the approach of comparing number of “copies in” to 

“copies out” by qPCR (Barta et al., 2014; Kemp et al., 2014) (Chapter 1). 

MATERIALS AND METHODS

Creation of the Pooled Standards 

Pooled standards containing the 16 fragments of genomic DNA amplified by the 

Promega PowerPlex 16® System (i.e., representing targeted DNA for mega-probe 

capture) and a 181 bp fragment of northern fur seal mitochondrial DNA (representing 

non-targeted DNA, and abbreviated “NSF mtDNA” throughout the remainder of the 

study) were created following Winters et (In Prep-a) (Chapter 2) to final concentrations of 

1 ×	105, 1 ×	104, and 1 ×	103 copies/μL. For example, the 1 ×	103 pooled standard 

contained 1000 copies of each of the sixteen PowerPlex 16® System amplicons and

1000 copies of NFS mtDNA per microliter.

Mega-probe Capture (Largely Following Maricic et al. (2010) (Figure 1) 

Preparation of the Mega-probe Bait 

To make double stranded Bio-T/B adaptors, Bio-T (5’-Biotin-TCAAGGACATCCG-3’) and 

Bio-B (5’-CGGATGTCCTTG-3’) were resuspended in a solution of 10 mM Tris and 10 

mM NaCl to 100 μM. Mixed together in equal portions (with final concentration of each 

primer at 50 μM), the solution was heated to 98°C for two minutes, then allowed to cool 

to room temperature on the bench top. 

A pooled standard containing 16 fragments of genomic DNA amplified by the Promega 

PowerPlex 16® System at a concentration of 1x107 copies/μL was made blunt ended with 

the Quick BluntingTM Kit (New England Biolabs®). The 100 μL reaction incubated for 30 

minutes at room temperature included: 1X Blunting Buffer, 0.1 mM dNTP mix, 4 μL 

Blunting Enzyme Mix, and 76 μL of the 1x107 standard DNA. This reaction was purified 

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82
using the Qiagen MinElute® PCR Purification Kit, following the manufacturer’s 

instructions except that the pH indicator was not added and final elution was performed 

in 15 μL of Buffer EBT (EB Buffer, 0.05% Tween-20). 

The double stranded Bio-T/B adaptors were ligated to the blunt-ended pooled DNA 

standard with the Quick LigationTM Kit (New England Biolabs®). The 40 μL reaction 

incubated at room temperature for 15 minutes included: 1X Quick Ligation Buffer, 1 μL of 

the Bio-T/B adaptors (50 μM), 4 μL of Quick T4 DNA Ligase, and the 15 μL of purified, 

blunt-ended pooled DNA standard. This reaction was purified using the Qiagen 

MinElute® PCR Purification Kit, following the manufacturer’s instructions except that the 

pH indicator was not added and final elution was performed in 15 μL of Buffer EBT 

Adding Mega-probe Bait to the Beads 

The 15 μL of prepared mega-probe bait was mixed with 15 μL of 2X BWT Buffer (2M 

NaCl, 10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 0.1% Tween-20). This mixture was

heated to 98°C for one minute and immediately placed on ice until further use. 

An aliquot of 50 μL of Dynabeads® M-270 Streptavidin-coated beads was prepared by 

placing them on the magnet (DynamagTM) for 2 minutes and removing the supernatant. 

An aliquot of 200 μL of 1X BWT Buffer was added to the beads. The mixture was 

vortexed and placed back on the magnet for 2 minutes and the supernatant removed. 

This was repeated twice more with volumes of 200 μL of 1X BWT Buffer added to the 

beads. Finally the beads were resuspended in 200 μL of TET Buffer [10 mM Tris-HCl 

(pH 8.0), 1mM EDTA (pH 8.0), 0.05% Tween-20]. 

The 30 μL of chilled, prepared mega-probe bait was added to the bead suspension. The 

tube was rotated for 20 minutes at room temperature, placed on the magnet for two 

minutes, and the supernatant removed. An aliquot of 200 μL of 1X BWT, preheated to 

50°C, was added, and then the tube was vortexed and placed back onto the magnet for 

two minutes. This was repeated once again. Finally the beads with immobilized megaprobes were resuspended in 50 μL of TET Buffer. 

Mega-probe capture of DNA Standards 

Three standards each at 1 ×	105, 1 ×	104, and 1 ×	103 copies/μL, as well as one negative 

control (dH2O) were subjected to mega-probe capture. A 26 μL aliquot of the pooled 

DNA standard was combined with 26 μL of Agilent hybridization buffer. This mixture 

was heated to 95°C for three minutes, then held at 37°C for 30 minutes. The tube was 

briefly centrifuged to spin down the liquid.

The tube containing bead-immobilized mega-probes was placed on the magnet for two 

minutes and the supernatant removed. The immobilized mega-probes were 

resuspended in the 52 μL of pooled DNA standard in Agilent hybridization buffer. The 

tube lid was secured with parafilm prior to incubation at 65°C for two nights. 

The tube was subsequently placed on the magnet for two minutes and the supernatant 

removed. An aliquot of 200 μL of 1X BWT Buffer was mixed with the captured DNA on 

the magnetic beads, the tube was placed back on the magnet for two minutes, and the 

supernatant removed. This was repeated twice more with 200 μL aliquots of 1X BWT 

Buffer. Preheated to 60°C, 200 μL of 1X HWT Buffer (2.5 mM MgCl2, 50 mM KCl, 15mM 

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83
 

Tris-HCl ph 8.0, 0.1% Tween-20) was added. The mixture was incubated at 60°C for 

two minutes, the tube placed back on the magnet for two minutes, and the supernatant 

removed. This was repeated again with the addition of 200 μL of 1X HWT Buffer 

preheated to 60°C. The captured DNA on the magnetic beads was washed once more 

in this fashion with 200 μL of 1X BWT. Finally the beads were resuspended in 100 μL of 

TET Buffer and this mixture was transferred to a new 1.5 mL tube. 

Dissociation of Captured DNA from Mega-probes 

The tube was placed on the magnet for two minutes and the supernatant discarded. An 

aliquot of 50 μL of 125 mM NaOH was added, the tube vortexed for ten seconds, and 

then placed back on the magnet for two minutes. The supernatant was transferred to a 

tube containing a neutralizing solution (250 μL of PB Buffer, 14 μL of 0.5 M HCl). An 

additional 50 μL aliquot of 125 mM NaOH was added to the beads, the tube vortexed for 

ten seconds, and then placed back on the magnet for two minutes. The supernatant 

was transferred to the same tube containing the neutralizing solution and an additional 

250 μL of PB Buffer and 14 μL of 0.5 M HCl were added. This mixture was purified 

using the Qiagen MinElute® PCR Purification Kit, following the manufacturer’s 

instructions except that the pH indicator was not added and final elution was performed 

in 15 μL of TET Buffer. The eluate volume was brought to 50 μL with dH2O. 

Hybridization Steps Only with Qiagen MinElute® PCR Purification Kit Purification 

In order to assess the influence on resulting copy number of the hybridization steps of

the mega-probe capture protocol, two 26 μL aliquots of each 1 ×	105, 1 ×	104, and 1 ×	

103 copies/μL pooled DNA standard were mixed with 26 μL of Agilent hybridization 

buffer heated to 95°C for three minutes, followed by a hold at 37°C for 30 minutes and 

then at 65°C for two nights. Two 26 μL aliquots each of the 1 ×	105 and 1 ×	104

copies/μL standard were mixed with 26 μL of Agilent hybridization buffer and subjected 

to the following heat treatments: 1) 95°C for three minutes, 2) 37°C for 30 minutes, or 3) 

65°C for two nights. All of these experimentally heat treated standards were purified by 

Qiagen MinElute® PCR Purification Kit, following the manufacturer’s instructions except 

that the pH indicator was not added and final elution was performed in 26 μL of dH2O.

Additional dH2O was added to bring the final volume to 50 μL. 

Quantitative PCR (qPCR) and Calculating Percent Retention and Loss 

The amount of Amelogenin, D13s317, D16s539, and NFS DNA remaining in the pooled 

DNA standard following experimental treatment (i.e., “copies out”) was determined by 

qPCR following Winters et al (In Prep-a) (Chapter 2). Quantification of each marker was 

conducted in duplicate against standard curves generated from two reactions each of 

standard pools at 1 ×	105, 1 ×	104, 5 ×	103, 2 ×	103, 1 ×	103, 5 ×	102, 2.5 ×	102, 1 ×	102

and 50 copies/μL. Two additional 1 ×	105, 1 ×	104, and 1 ×	103 copies/μL standards 

(untreated) each were quantified as unknowns to monitor the concentration of preexperimental standards. These provide estimates of “copies in”. 

Subtraction of the number of “copies out” (measured as the average of duplicate qPCR 

amplifications from the molecules retained following purification) from “copies in” 

(measured as the average of duplicate qPCR amplifications of the standards treated as 

unknowns) divided by “copies in” multiplied by 100 provides the percent retention:

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84
 

[(copies in - copies out)/ copies in] ×	100 

One hundred minus retention provides a measure of percent loss. Retention and loss of 

Amelogenin, D13s317, D16s539, and NFS mtDNA was determined in this manner three 

times, from which the average loss and the uncorrected sample standard deviation (i.e., 

the standard deviation of the sample) were calculated. 

Statistical Analyses 

Two-tailed t-tests were used to statistically evaluate differences in mean percentage 

retention at the 0.05 level of probability. Statistical analyses were conducted in StatPlus 

(AnalystSoft Inc.).

RESULTS 

Quantitative PCR efficiencies for amelogenin, D13s317, and D16s539 ranged from 

91.20-97.10%, with R2 values ranging 0.9984-0.9994 (Tables 1-5), comparable to 

previous observations (Kemp et al., 2014; Winters et al., In Prep-b; Winters et al., In 

Prep-a) (Chapters 1-3). The NSF mtDNA qPCR efficiencies ranged from 87.48-92.04% 

with R2 values from 0.9987-0.9993, also comparable to previous observations (Winters 

et al., In Prep-b; Winters et al., In Prep-a) (Chapters 2 & 3).

Across all three standards (1 ×	105, 1 ×	104, and 1 ×	103 copies/μL), average DNA 

retention of targeted amelogenin, D13s317, and D16s539 DNA following mega-probe 

capture was 702.46%, with tremendously variable retention percentages across markers 

and the standards (Table 1). This was likewise true of the non-targeted NFS mtDNA, 

with an average retention of 307.04%. 

An average of 83.04% of the DNA was lost from the purified standards (1 ×	105, 1 ×	104, 

and 1 ×	103 copies/μL) that were heated to 95°C for three minutes, followed by a hold at 

37°C for 30 minutes and then at 65°C for two nights. An average of 51.31% of the DNA 

was lost from the purified pooled DNA standards (1 ×	105 and 1 ×	104 copies/μL) that 

were heated to 95°C for three minutes. An average of 63.94% and 68.25% of the DNA 

was lost from the same purified standards following subjection to 37°C for 30 minutes 

and 65°C for two nights, respectively. 

DISCUSSION 

In this study we sought to document the efficiency of mega-probe capture (Figure 1) as a 

method to enrich DNA extracts by two measures, first in its ability to retain targeted DNA 

molecules and secondly in its ability to remove non-target DNA molecules. However, 

unlike our previous efforts to measure the efficiency of fishing and PEC by these same 

measures (Winters et al., In Prep-b; Winters et al., In Prep-a) (Chapters 2 & 3) the 

results are not as straightforward to interpret. Instead of being less than 100% efficient 

at retaining target DNA molecules, our observations reveal an average 702.46% 

increase in these molecules. This was an unexpected outcome, one that is impossible 

and, thus, demands some explanation. One clue is that our results demonstrate that the 

absolute number of copies retained across each targeted DNA fragment is relatively 

consistent (Table 1). For example, across the amelogenin standards that span three 

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85
orders of magnitude in copy number (1 ×	103 to 1 ×	105 copies/μL), the number of 

retained target molecules differ only by ~11000 molecules (29610.26-50603.96). It is 

conceivable that a portion of the bait molecules became disassociated from the 

streptavidin-coated beads, losing their status as bait and, thus, gaining the ability to be

counted as part of the pooled DNA standard (i.e., inadvertently adding to the number of 

copies out). This idea is supported by our observation of 6217.43, 14555.55, and 

36621.00 copies/μL of D16s539, D13s317, and amelogenin, respectively, in the megaprobe negative control [bait molecules and dH2O (Table 1)]. 

More difficult to explain is our observation of 5169.17 copies/μL of NSF mtDNA in the 

mega-probe negative control, given that it contained neither NSF mtDNA nor NSF 

mtDNA mega-probes to capture them. Looking again at the dissociation curve for these 

reactions, nothing abnormal was observed. One likely explanation is that the incorrect 1 

×	107 standard was used to create the mega-probe bait, one that contained 1 ×	107 

copies/μL of NSF mtDNA. If this is the case, we would not be able to draw conclusions 

about the efficiency of mega-probe capture in removing non-target molecules. However, 

observing NSF mtDNA in a negative control is consistent with the notion of bait 

molecules being transformed into countable molecules. While follow up experiments are 

warranted to account for this, overall the results reported here, combined with megaprobe capture being a relatively cumbersome and time intensive method, we did not do 

so. 

In our previous studies aimed to evaluate the efficiency of fishing for DNA (Winters et al., 

In Prep-a)(Chapter 2) and PEC (Winters et al., In Prep-b)(Chapter 3), we were able to 

identify some of the steps within the respective protocols that contribute to their 

inefficiencies. Specifically, we demonstrated that 61.35-69.49% of the molecules are

“lost” during the essential hybridization step of the fishing protocol and that 56.69% of 

the molecules are “lost” following extension and purification of the extended products, 

steps that are essential steps to the PEC protocol. Since this loss of molecules follows 

heat treatment of the DNA, it is likely that the molecules are losing one or both priming 

regions, leading to them being non-amplifiable and, thus, not quantified during qPCR 

(Winters et al., In Prep-b; Winters et al., In Prep-a). 

During the current study we observed losses of DNA molecules following subjection

specifically to the hybridization steps employed in the mega-probe protocol. An average 

of 83.04% of the DNA was lost following subjection to 95°C for three minutes, followed 

by a hold at 37°C for 30 minutes and then at 65°C for two nights. Subjection to each of 

these hybridization steps separately resulted in 51.31%, 63.94%, and 68.25% loss of the 

DNA molecules, respectively. There appears to be a positive relationship between 

increasing DNA loss and time exposure to heat. While the accumulation of DNA 

damage with heat exposure is an expected outcome (Lindahl, 1993), the magnitude of 

loss was quite surprising to us. It is important to keep in mind that some of the DNA is 

lost following purification with the Qiagen MinElute® PCR Purification Kit (Kemp et al., 

2014) (Chapter 1) and, thus, a correction factor may be applied to gain a better 

perspective on DNA loss due to the heat treatment alone. As an example, we observed 

an average of 80.87% loss of amelogenin DNA from the 1 ×	105 standard, resulting in 

and outcome of 21044.19 copies (Table 2). Since we can expect to lose 52% of these 

molecules during purification (Kemp et al., 2014) (Chapter 1), prior to purification we 

could estimate the copy number to have been ~40469.60. Thus, heat treatment of 95°C 

for three minutes, followed by a hold at 37°C for 30 minutes and then at 65°C for two

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nights is responsible for ~63% of the loss. It is critically important for those working with 

LCN and degraded DNA samples to be mindful of the loss of already limited DNA 

strands available for analysis as a result of the experimental treatments used to study 

them. 

While mega-probe is an established method for enriching target DNA from libraries prior 

to HTS (Maricic et al., 2010; Reich et al., 2010), our experiments raise concern over its 

use as a DNA extraction method, despite some of our own methodological problems in 

evaluating its efficiency. Of principal concern is the possibility that the mega-probe bait 

becomes quantified as captured target molecules, and may in turn lead to the 

misrepresentation of the endogenous molecules in downstream applications. 

ACKNOWLEDGMENTS 

Thanks to Kathleen Judd for her assistance in the laboratory. 

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87
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ancient DNA. Forensic Sci Int Genet 2:104-7. 

Barta JL, Monroe C, Teisberg JE, Winters M, Flanigan K, and Kemp BM (2014) One of 

the Key Characteristics of Ancient and Forensic DNA, Low Copy Number, May 

be a Product of its Extraction. Journal of Archaeological Science 46:281-289. 

Briggs AW, Good JM, Green RE, Krause J, Maricic T, Stenzel U, Lalueza-Fox C, Rudan 

P, Brajkovic D, Kucan Z, Gusic I, Schmitz R, Doronichev VB, Golovanova LV, de 

la Rasilla M, Fortea J, Rosas A, and Paabo S (2009) Targeted retrieval and 

analysis of five Neandertal mtDNA genomes. Science 325:318-21. 

Carpenter ML, Buenrostro JD, Valdiosera C, Schroeder H, Allentoft ME, Sikora M, 

Rasmussen M, Gravel S, Guillén S, Nekhrizov G, Leshtakov K, Dimitrova D, 

Theodossiev N, Pettener D, Luiselli D, Sandoval K, Moreno-Estrada A, Li Y, 

Wang J, Gilbert MTP, Willerslev E, Greenleaf WJ, and Bustamante CD (2013) 

Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of 

Ancient DNA Sequencing Libraries. American Journal of Human Genetics 

93:852–864.

Kemp BM, Winters M, Monroe C, and Barta JL (2014) How Much DNA is Lost? 

Measuring DNA Loss of Short-Tandem-Repeat Length Fragments Targeted by 

the PowerPlex 16® System Using the Qiagen MinElute Purification Kit. Human 

Biology 86:313-329. 

Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362:709-

715. 

Maricic T, Whitten M, and Paabo S (2010) Multiplexed DNA sequence capture of 

mitochondrial genomes using PCR products. PLoS One 5:e14004. 

Nandineni MR, and Vedanayagam JP (2009) Seletive enrichment of human DNA from 

non-human DNAs for DNA typing of decomposed skeletal remains. Forensic Sci 

Int Genet 2:520-521. 

Reich D, Green RE, Kircher M, Krause J, Patterson N, Durand EY, Viola B, Briggs AW, 

Stenzel U, Johnson PL, Maricic T, Good JM, Marques-Bonet T, Alkan C, Fu Q, 

Mallick S, Li H, Meyer M, Eichler EE, Stoneking M, Richards M, Talamo S, 

Shunkov MV, Derevianko AP, Hublin JJ, Kelso J, Slatkin M, and Paabo S (2010) 

Genetic history of an archaic hominin group from Denisova Cave in Siberia. 

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Wang J, and McCord B (2011) The application of magnetic bead hybridization for the 

recovery and STR amplification of degraded and inhibited forensic DNA. 

Electrophoresis 32:1631-8. 

Winters M, Monroe C, Barta JL, and Kemp BM (In Prep-a) Are We Fishing or Catching? 

Evaluating the Efficiency of Bait Capture of CODIS Fragments.

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Capture as a Method to Enrich DNA Extractions. 

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Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

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Table 1. Retention of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mt

following megaprobe capture.

Capture 1 Capture 2 Capture 3 Average 

% 

Retention 

 E

R2 Copies In SD 

Copies 

Out SD 

Copies 

Out SD 

Copies 

Out SD 

Copies 

Out SD 

Amelogenin 95.97 0.9994

109074.00 1153.37 35275.19 422.63 20444.13 18.90 33111.44 591.93 29610.26 8011.48 27.15 

10889.20 961.00 68499.62 129.73 15717.47 79.73 67594.81 2407.43 50603.96 30215.98 464.72 

952.09 5.27 37968.27 1453.65 27014.13 213.08 25460.38 772.00 30147.60 6817.31 3166.47 

D13s317 93.04 0.9992

88395.50 1752.54 14577.77 545.29 6364.17 1621.97 13784.85 n/a 11575.60 4530.60 13.10 

8022.76 245.00 25185.58 n/a 6201.13 n/a 26388.27 584.18 19258.33 11323.84 240.05 

637.19 18.00 12428.33 1217.91 10185.25 197.66 9059.19 n/a 10557.59 1715.15 1656.91 

D16s539 91.20 0.9993

112950.00 1625.65 8847.77 244.79 4049.19 4.98 8407.57 351.80 7101.51 2652.53 6.29 

11560.80 1396.11 16256.90 382.19 3240.25 201.63 15003.00 54.12 11500.05 7180.62 99.47 

991.58 51.40 7209.66 173.25 6724.84 71.70 5342.47 76.90 6425.66 968.88 648.02 

Average 702.46 

NFS mtDNA 87.48 0.9993

84906.70 n/a 7722.26 255.93 4183.27 n/a 8235.33 n/a 6713.62 2206.31 7.91 

9264.85 299.00 13962.54 n/a 2818.17 n/a 11968.96 726.63 9583.22 5942.90 103.44 

727.21 74.90 7018.73 n/a 5996.15 3.15 4651.54 n/a 5888.81 1187.24 809.79 

Average 307.04 

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Table 2. Loss of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mtDNA

by Qiagen MinElute® PCR Purification Kit following subjection to 95°C for three minutes, followed by a 

minutes and then at 65°C for two nights.

Standard 1 Standard 2 Average

% Loss

E

R2 Copies In* SD Copies Out SD Copies Out SD Copies Out SD 

Amelogenin 94.97 0.9991

109491.00 2601.30 13026.08 682.74 29062.31 744.09 21044.19 11339.33 80.78

10445.10 1410.22 1624.72 22.54 1394.12 16.76 1509.42 163.06 85.55

1165.84 30.50 396.61 54.45 87.54 4.12 242.07 218.55 79.24

D13s317 94.39 0.9990

93568.80 13842.40 15503.57 249.65 26938.08 n/a 21220.82 8085.42 77.32

9239.72 609.00 1498.68 n/a 1492.54 n/a 1495.61 4.34 83.81

861.66 26.30 343.61 n/a 90.30 n/a 216.96 179.11 74.82

D16s539 92.01 0.9991

128737.00 3534.84 12630.38 532.07 21645.19 3273.09 17137.79 6374.43 86.69

12879.10 485.00 1221.24 21.18 1080.24 47.40 1150.74 99.70 91.07

1302.15 131.00 293.94 8.85 77.91 6.23 185.92 152.76 85.72

NFS mtDNA 88.09 0.9987

79412.90 15408.50 11002.62 195.65 19855.87 330.84 15429.24 6260.19 80.57

9810.36 n/a 1344.97 n/a 1221.51 n/a 1283.24 87.30 86.92

918.90 90.20 234.05 77.57 60.88 10.86 147.47 122.45 83.95

Average 83.04

*values for 1X105 and 1X104 copies in are associated with efficiencies (E) and coefficients of determination (R2) found in Tables 3 and 4

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
Table 3. Loss of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mtDNA

by Qiagen MinElute® PCR Purification Kit following subjection to 95°C for three minutes. 

Standard 1 Standard 2 Average

% Loss

E

R2 Copies In SD Copies Out SD Copies Out SD Copies Out SD 

Amelogenin 96.50 0.9990 109491.00 2601.30 53315.58 2397.09 54138.27 748.99 53726.92 581.73 50.93 10445.10 1410.22 4120.40 305.39 5616.32 233.96 4868.36 1057.77 53.39D13s317 94.80 0.9984 93568.80 13842.40 52795.67 1786.94 54998.27 1070.45 53896.97 1557.47 42.40 9239.72 609.00 4511.63 n/a 5496.54 n/a 5004.09 696.43 45.84D16s539 97.10 0.9990 128737.00 3534.84 58365.87 458.40 63959.71 1931.63 61162.79 3955.45 52.49 12879.10 485.00 4558.87 337.43 5975.36 201.62 5267.11 1001.61 59.10NFS mtDNA 92.04 0.9988 79412.90 15408.50 39985.77 818.88 41285.58 4472.99 40635.67 919.10 48.83 9810.36 n/a 3734.65 n/a 4612.77 n/a 4173.71 620.92 57.46

Average 51.31

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
Table 4. Loss of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mtDNA

by Qiagen MinElute PCR Purification Kit following subjection to 37°C for 30 minutes. 

Standard 1 Standard 2 Average

% Loss

E

R2 Copies In SD Copies Out SD Copies Out SD Copies Out SD 

Amelogenin 96.50 0.9990 109491.00 2601.30 45645.48 2859.84 42972.12 5295.14 44308.80 1890.35 59.53 10445.10 1410.22 2429.00 329.43 3071.35 30.51 2750.17 454.21 73.67D13s317 94.80 0.9984 93568.80 13842.40 44799.23 n/a 41970.38 n/a 43384.81 2000.30 53.63 9239.72 609.00 2963.44 208.00 3250.19 34.29 3106.82 202.76 66.38 D16s539 97.10 0.9990 128737.00 3534.84 54235.67 150.80 51800.19 1462.62 53017.93 1722.14 58.82 12879.10 485.00 3346.28 319.79 4052.28 7.14 3699.28 499.22 71.28 NFS mtDNA 92.04 0.9988 79412.90 15408.50 38022.12 n/a 34478.08 n/a 36250.10 2506.01 54.35 9810.36 n/a 2382.46 49.80 2739.90 382.30 2561.18 252.75 73.89

Average 63.94

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
Table 5. Loss of Amelogenin, D13s317, D16s539, and northern fur seal mitochondrial DNA (NSF mtDNA

by Qiagen MinElute® PCR Purification Kit following subjection to 65°C for two nights. 

Standard 1 Standard 2 Average

% Loss

E

R2 Copies In SD Copies Out SD Copies Out SD Copies Out SD 

Amelogenin 94.97 0.9991 109491.00 2601.30 25622.60 341.99 40484.52 1928.91 33053.56 10508.97 69.81 10445.10 1410.22 3285.85 78.41 3650.63 389.63 3468.24 257.94 66.80D13s317 94.39 0.9990 93568.80 13842.40 24188.56 424.67 37541.54 n/a 30865.05 9441.98 67.01 9239.72 609.00 2870.63 150.26 3821.10 485.51 3345.87 672.08 63.79D16s539 92.01 0.9991 128737.00 3534.84 27149.04 83.22 45039.71 2301.22 36094.38 12650.62 71.96 12879.10 485.00 3546.63 136.35 4735.40 105.90 4141.01 840.59 67.85NFS mtDNA 88.09 0.9987 79412.90 15408.50 20007.69 923.05 31290.58 n/a 25649.13 7978.20 67.70 9810.36 n/a 2484.29 14.88 3192.18 551.94 2838.24 500.56 71.07

Average 68.25

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.
Figure 1. Simplified Schematic of Mega-Probe Capture [largely following Maricic 

et al (2010)]. Target DNA molecules are green (in this study, representing the 16 

fragments targeted by PowerPlex 16® System), non-target molecules are red (in this 

study, representing NFS mtDNA), biotinylated mega-probes are blue, and the 

streptavidin coated magnetic beads are white. 

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

94
DISSEMINATION OF RESEARCH FINDINGS

Publications 

2014 Kemp BM, Winters M, Monroe C, and Barta JL. “How Much DNA is Lost? 

Measuring DNA Loss of STR Length Fragments Targeted by the PowerPlex 16® 

System using the Qiagen MinElute® Purification Kit.” Human Biology 86(4): 313-

329.

This resource was prepared by the author(s) using Federal funds provided by the U.S. 

Department of Justice. Opinions or points of view expressed are those of the author(s) and do not 

necessarily reflect the official position or policies of the U.S. Department of Justice.

95