Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Genomic DNA Isolation from Amplified

Product for Recursive Amplification of

Low-Template DNA Samples

Joseph R. Iacona, M.S.

Amy N. Brodeur, M.F.S.

Catherine M. Grgicak, Ph.D.

Introduction

Forensic laboratories continuously struggle with low-template

DNA analysis

Currently, there are three approaches labs have taken to improve

low-template analysis:

Protocol adjustments such as changing the master mix,

using post-PCR purifications, decreasing the analytical

threshold, etc.1

Consensus profiling – splitting the extract between three

(or more amplifications); if a peak is observed at least twice,

then it is designated as a true allele2

Re-amplification – the act of amplifying an aliquot of the

PCR product

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Introduction

However, these approaches have been plagued with problems

Gill et al. have suggested that consensus profiling may be

ineffective for extreme low-template samples (<100 pg) due to

high frequency of stutter and stochastic effects3,4

Attempts at re-amplification showed issues

such as aborted PCR and smeared

gel electrophoresis bands5

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Lou R, DaMing Z. Partial strands synthesizing leads to inevitable aborting and complicated

products in consecutive polymerase chain reactions (PCRs). Science in China Series C 2007

50:546-56.

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Recursive Amplification Schematic

1 3 2 4

Shea J, Streptavidin-Biotin Binding of DNA Amplicons: Methods for the Typing

and Re-typing of Forensically Relevant Short Tandem Repeats (Master’s

Thesis). 2012, Boston University School of Medicine, Boston, MA.

Non-optimal:

• Some amplicons are not removed

by the streptavidin beads

• Primers and other PCR

components are not completely

filtered out

• Template DNA is lost during either

step of the process

Goals of the Study

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Continue development of recursive amplification technique,

specifically focusing on the post-PCR purification methods

Assess the percent recovery of original template DNA

Assess the success of amplicon removal by streptavidin-coated

beads

Determine ways to improve above mentioned factors in order to

increase usefulness of the method for forensic purposes

Methods: Method Run-Through

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Used to calculate percent

recovery:

% recovery =

mass of “cleaned”

DNA/input DNA mass x

100%

Results: Assessing Method

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Leftover

TPOX

Signal

Successful

Re-amp

Average D5S818

Peak Height =

4073 ± 2500 RFU

Average Retained

TPOX Peak Height

= 1122 ± 1050 RFU

Results: Percent Recovery

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Average Percent Recovery = 37 ± 28%

Average Percent Loss = ~63%

Benefits of using

qPCR:

• Show the

successful re-

amplification of a

second locus

(RPPH1)

• Allows for more

accurate percent

recovery

calculations

Results: Dynamic Model

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

A computer-based dynamic model was created to represent the

process as a constantly changing system with cause-and-effect

relationships

The model was designed such that:

it could be used to obtain peak heights without physically

running an experiment in the laboratory,

total genomic DNA loss could be estimated given the

resulting peak heights,

total signal retention could also be estimated,

and it could be used as a tool during method optimization.

Given the current average peak heights, the model suggested an

approximate genomic DNA loss of 68%

Results: Percent Recovery

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

If 60-70% of the template DNA is lost during the post-

PCR purification steps:

• During which step(s) is the loss occurring?

• How much is lost during each step?

• What is causing this to occur?

Results: Bead Removal of Template DNA

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Average Mass of Template DNA on Beads = 0.24 ± 0.12 ng

Average Percent Loss on Beads = 11 ± 6%

Potential cause: Biotinylated primers hybridized to template DNA are

causing the DNA to be pulled out during the bead step.

Results: Loss of Template DNA in Filter

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Average Percent Loss in Filter = 66 ± 12%

The filtration step therefore contributes to most of

the template DNA loss.

Potential cause: High speed of filtration spin (i.e. 14,000 rcf) causes

the template DNA to become stuck in the filter membrane6.

Results: Retained TPOX Peak

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Amplicons not

removed by beads

(8,8 position)

No amplification from

retained primers

(11,11 position)

DNA 1 8,8 genotype TPOX Amp - DNA 1 Cleaned samples Added DNA 2 11,11 genotype No primers added TPOX Amp 8 peak retained amplicons 11 peak retained primers 8 & 11 both factors

Optimization: Spin Speed Analysis

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Comparing:

p value

t value

Significant?

14,000 and 4,000 rcf

0.0101 4.5945 Yes

14,000 and 3,500 rcf

0.0228 3.5984 Yes

14,000 and 3,000 rcf

0.4094 0.9204 No

14,000 and 2,500 rcf

0.0199 3.7535 Yes

4,000 and 3,500 rcf

0.6085 0.555 No

Conclusion: Performing the filtration spin at 4,000

– 3,500 rcf leads to a statistically significantly higher

percent recovery from the current protocol, with a

30-40% increase in recovery.

Optimization: Concentration Spin Analysis

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

Comparing:

p value

t value

Significant?

1,000 and 5,000 rcf

0.7467

0.3461

No

1,000 and 10,000 rcf

0.7529

0.3372

No

1,000 and 14,000 rcf

0.3281

1.113

No

Conclusion: Performing the concentration spin at

higher speeds does not lead to a statistically

significantly higher percent recovery from the

current protocol.

Conclusions

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

• The concept of recursive amplification is a viable one

• A significant amount of DNA was lost during the cleanup

• Retained signal from the original amplification remains was

detected in cleaned samples

• Approximately 20% of the observed total template DNA loss is

due to removal by the beads, while the rest is from the filtration

step, possibly from becoming caught within the membrane6

• The retained amplicon signal is due to insufficient bead cleaning

and not from primers remaining in solution after filtration

• In general, lowering the spin speed of the filtration improves the

percent recovery

Acknowledgements

• Boston University School of Medicine Biomedical Forensic

Sciences Program

• Supported by Northeastern Association of Forensic

Scientists Carol DeForest Forensic Science Student

Research Grant

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

References

Boston University School of Medicine

Program in Biomedical Forensic Sciences

72 E. Concord Street, Boston, MA 02118

1. Smith P, Ballantyne J. Simplified low copy number DNA analysis by

post-PCR purification. J. Forensic Sci. 2007 52:820-9.

2. Grisedale KS, Daal Av. Comparison of STR profiling from low template

DNA extracts with and without the consensus profiling method. 2012

3. Bright JA, Gill P, Buckleton J. Composite profiles in DNA analysis.

Forensic Sci. Int.- Genetics 2012 6:317-21.

4. P. Gill, J.P. Whitaker, C. Flaxman, N. Brown, J.S. Buckleton, An

investigation of the rigor of interpretation rules for STR’s derived from

less that 100 pg of DNA, Forensic Sci. Int. 112 (1) (2000) 17–40.

5. Lou R, DaMing Z. Partial strands synthesizing leads to inevitable

aborting and complicated products in consecutive polymerase chain

reactions (PCRs). Science in China Series C 2007 50:546-56.

6. Garvin AM, Fritsch A, Purifying and Concentrating Genomic DNA from

Mock Forensic Samples using Millipore Amicon® Filters. J. Forensic

Sci. 2013 58:S173-S4.