Applied 

Genetics

Exploring DNA Extraction 

Efficiency

Erica Butts

Research Biologist, Applied Genetics Group

Forensics@NIST 2012 Meeting

Gaithersburg, MD

November 28, 2012
Applied 

Genetics Outline

• Define DNA extraction 

• Downstream concerns for DNA extraction

• Methods used to evaluate extraction efficiencies

• Define absolute extraction efficiency

• What can we learn from absolute extraction 

efficiency?

• Alternate approaches to extraction
Applied 

Genetics DNA Extraction

Cell Lysis

(Break open the 

cell to release 

the proteins that 

protect the 

DNA)

Removal of 

Lipids 

(By adding a 

detergent)

Removal of 

Proteins

(By adding a 

protease)

DNA Binding

(DNA Binds to 

magnetic or silica 

beads and is 

washed)

Precipitation of 

DNA

(DNA falls out of 

solution)

Rehydration of 

DNA

(Adding desired 

buffer)

Elution of DNA

(Into desired buffer)

DNA extraction is the first step after collection in the DNA typing process

Purification methods are often used to try to eliminate the presence of additional 

proteins, lipids, and inhibitors
Applied 

Genetics Challenges of DNA Extraction

• Organic extraction processes (manual) involves 

hazardous chemicals such as phenol and 

chloroform

• Liquid handling steps may increase risk for 

contamination and loss of sample 

– Employing robotics can decrease this challenge

• Inhibitors found in the original sample may be 

carried through the extraction process

– Inhibitors may reduce the PCR efficiency of assays being 

used

• Two common PCR inhibitors found in forensic 

samples

– Hemoglobin (Blood)

– Indigo dye (Denim)
Applied 

Genetics Definition of Relative Extraction Efficiency

• Recovery compared to another method of extraction (often 

organic)

• Defined using several different metrics

– Full vs. Partial STR Profiles

– Number of loci successfully genotyped (percent of profile obtained)

Method A

Method B

Loci Called: 5/5

Profile: 100%

Loci Called: 3/5

Profile: 60%
Applied 

Genetics Limitations of Relative Efficiency 

Metrics

• Measures end point of genotyping process

– Efficiency of STR genotyping

• Does not reflect the absolute efficiency of only 

the extraction process 

• Does not account for the initial amount DNA 

present in the sample

– In forensic samples the true amount of starting 

material is unknown due to the source of the sample
Applied 

Genetics Absolute Extraction Efficiency

• The ratio of the amount of DNA recovered 

(quantitated) to the original amount of DNA 

(known) after extraction

• This offers the ability to evaluate the absolute 

efficiency of the extraction

• The original amount needs to be known
Applied 

Genetics Testing Absolute Extraction Efficiency

Highly characterized extracted DNA: Varying amounts 

added to sterile swabs (n=18 per quantity)

Known quant value: 52.44 ng/μL

Ranges from 1500 ng to 100 ng

Human epithelial cell lines*: 100 μL of a cell suspension 

swabbed from Teflon tube (n=12 per quantity)

Number of cells determined through flow cytometry

Ranges from 1200 ng to 300 ng of DNA

Whole blood*: Seven volumes of whole blood tested (n=2 per 

volume)

Ranges from 4800 ng to 24 ng of DNA

*Assume 6 pg of DNA per cell

Placing a known amount of DNA into the extraction process and 

determine the amount recovered

DNA Sources:
Applied 

Genetics Extraction Methods

Qiagen EZ1 Advanced XL

• Swabs & Blood Stains 

– Pretreated with a Pro K digest 

and G2 Buffer at 56 C for 15 

minutes

– Pro K inactivation at 95 C for 

5 minutes

• Whole Blood

– Total sample volume brought 

up to 200 μL with G2 Buffer

• Eluted with 100 μL TE 

Modified Salt Out

• Manual extraction process

• Involves a Proteinase K 

digest

• Saturated Ammonium 

Acetate solution to separate 

DNA

• Absolute Ethanol wash to 

precipitate DNA

• Rehydrated with 100 μL TE 
Applied 

Genetics Blood Extracted DNA Cells

Extraction Efficiency Across All Samples

n=14 n=18 n=12

Absolute extraction efficiency average: 16% 7%
Applied 

Genetics Summary of Absolute Extraction 

Efficiency

• Our experiments: 16% 7% average absolute 

extraction efficiency (Range from 4% to 41%)

• Literature studies: 16-33% absolute extraction 

efficiency

– A. Colussi et al. “Efficiency of DNA IQ System in recovering semen from cotton swab.” Forensic Science International: Genetics 

Supplement Series 2 (2009) 87-88.

– R. Kishore et al. “Optimization of DNA Extraction from Low-Yield and Degraded Samples Using the BioRobot EZ1 ad BioRobot M48.” J 

Forensic Sci, September 2006, Vol. 51, No 5.

– Y.C. Swaran, L. Welch, “A comparison between direct PCR and extraction to generate DNA profiles from samples retreived from 

various substrates.” Forensic Sci. Int. Genetics. May 2012, Vol. 6, No. 3.

• Loss of about 70-85% of initial sample during 

the extraction process

• Loss is independent of extraction method or 

source of DNA (i.e. blood, cells, previously 

extracted)
Applied 

Genetics Why Does This Matter?

• A majority of sample is lost during extraction

– Minimal impact on reference samples

– Enough DNA is recovered for an STR profile

• Low extraction efficiency could result in lower 

sample quantity which may fail to yield full STR 

profiles

1 ng

~ 70-80% sample loss

Extraction process

200 - 300 pg

May Result in Partial 

STR Profile
Applied 

Genetics

Alternate Extraction Techniques 

& 

Direct PCR

Is it possible to bypass 

extraction?
Applied 

Genetics Direct PCR

• Direct PCR kits commercially available

– Improved polymerase/master mix help limit inhibition

– Eliminates the need for purification

– Higher sensitivity

– Optimized for samples on FTA Cards

• Pretreatment protocols for other substrates 

Collection

Extraction Quantitation

Data

Interpretation

PCR Amplification

Separation/

Detection

Direct PCR Amplification
Applied 

Genetics Alternate Techniques for Direct PCR

• Buccal Swab Pretreatment for Direct PCR

– Prep-N-Go Solution (Life Technologies)

– SwabSolution Reagent (Promega)

• Blood Stains on non-FTA paper Pretreatment for 

Direct PCR

– Prep-N-Go Solution (Life Technologies)

– PunchSolution Reagent (Promega)

Pretreatment steps aid in breaking open the cell to lyse 

the DNA without purification
Applied 

Genetics Buccal Swab Pretreatment

Prep-N-Go Solution

• Incubate swab at room 

temperature in 400 μL 

Prep-n-Go Buffer

• 3 μL extract solution 

added directly to PCR

SwabSolution Reagent 

• Incubate swab at 70 °C 

for 30 minutes in 1 mL 

SwabSolution Reagent 

• 2 μL extract solution 

added directly to PCR

Life Technologies: GlobalFiler Promega: PowerPlex Fusion
Applied 

Genetics Blood Stains (non-FTA) Pretreatment

Prep-N-Go Solution

• 3 μL added with PCR 

setup and one 1.2 mm 

punch

PunchSolution Reagent 

• 10 μL PunchSolution

Reagent incubated at 70 °C 

for 30 minutes until punches 

are dry

Life Technologies: GlobalFiler

Promega: PowerPlex Fusion
Applied 

Genetics Overall Conclusions

Absolute Extraction Efficiency

• 16% 7% recovery yield when evaluating absolute 

extraction efficiency

– Independent of extraction method or DNA source

• Extraction chemistries could be optimized to 

increase yield

Direct PCR

• Direct PCR with pretreatment applications are an 

effective way to bypass low extraction 

efficiencies for reference samples.

– The need for a quantitation step prevents casework from 

applying direct PCR techniques

– Complete STR profiles can be generated from non-FTA 

substrates
Applied 

Genetics Acknowledgments

Contact Info: 

erica.butts@nist.gov

301-975-5107

Pete Vallone

Funding from the FBI 

Biometrics Center of 

Excellence ‘Forensic 

DNA Typing as a 

Biometric Tool’ 

NIST Team for This Work

Funding from the 

National Institute of 

Justice (NIJ) through 

NIST Office of Law 

Enforcement Standards

Margaret Kline

NIST Disclaimer: Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as 

completely as possible. In no case does such identification imply a recommendation or it imply that any of the materials, instruments or 

equipment identified are necessarily the best available for the purpose.

Points of view are those of the presenters and do not necessarily represent the official position of the National Institute of Standards and 

Technology or the U.S. Department of Justice.
Applied 

Genetics Thank you for your attention!