Applied 

Genetics

Stability Studies for DNA 

in Bloodstains and as 

Extracted Material.

Margaret Kline

Research Biologist, Applied Genetics Group

Forensics@NIST 2012 Meeting

Gaithersburg, MD

November 28, 2012
Applied 

Genetics Why do we care about the 

stability of DNA?

There’s two sides of that coin

Find the guilty

Protect the innocent
Applied 

Genetics

55 years
Applied 

Genetics
Applied 

Genetics Considerations for DNA Stability studies?

• What is the form of your DNA?

–Bloodstain or Buccal swab

–Extracted liquid in a tube

–Extracted and dried in a tube or as a stain

–Stabilized with a matrix/additive
Applied 

Genetics Considerations for DNA stability studies?

• What are the best conditions for storage?

– Room temperature?

– Refrigerated?

– Frozen?

• How long you can keep a sample under 

specified storage conditions?

– Days?

– Months?

– Years?
Applied 

Genetics Considerations for DNA stability studies?

• What environmental factors influence stability?

– Temperature excursions

• Power failures

• Shipping/ Transporting the samples

• Does the sample need to stay pristine, or can 

you tolerate some degradation?

• What are you measuring and how do you rate 

the results, DNA Quality/Quantity or both?
Applied 

Genetics So what metric are you going to use?

0 500 1000 1500 2000 2500

0 500 1000 1500 2000 2500

Allele 2, RFU

Allele 1, RFU

Peak Height Summary

Since our bottom line is can you get an STR profile ?

We can take the electropherogram and plot allele1 vs allele 2 peak heights

Or the results of matrix 1 vs matrix 2
Applied 

Genetics Dried Bloodstains

• Blood from 2 different donors

• Storage Media

FTA paper

903 paper

• Environmental factors

37 C (some places this is ambient)

Laboratory Room Temperature

-20 C.
Applied 

Genetics DNA/Bloodstain Storage Papers

903

• high-purity cotton linter 

pulp 

• no chemicals added

• DNA not bound to paper

FTA

• high-purity cotton linter 

pulp

• chemically treated with 

several compounds 

designed to kill 

pathogens and resist 

bacterial growth and DNA 

degradation Tris, EDTA, 

SDS, and uric acid

• DNA binds to paper
Applied 

Genetics Quality of the Extracted DNA

250 bp

1500 bp

L 1 2 3 4 5 6 7 8 9 10 11 12

L ladder with 250 bp, 400 bp, 800 bp and 1500 bp bands visible

Lanes 1, 2: + 37 C FTA; Lanes 3, 4: + 37 °C 903; 

Lanes 5, 6: RT FTA; Lanes 7, 8: RT 903;

Lanes 9, 10: -20 C FTA; Lanes 11, 12: -20 °C 903; 

After 11 years of storage at 37 C both FTA and 903 show signs of degradation, 

the FTA samples exhibit DNA with slightly higher molecular weight than the 903 

samples.

100 bp

+37 C RT -20 C

FTA 903 FTA 903 FTA 903

Most STR 

typing kits have 

products that 

are less than 

450 bp

450 bp
Applied 

Genetics FTA: 903 +37 C Storage

Normal length amplicon typing kit

Allelic dropout

CSF

D2S1338 D13S317

FTA

903
Applied 

Genetics 903 +37 C Storage 

Minimum length amplicon typing kit

D13S317

D2S1338

CSF

Alleles dropping out before are recovered.
Applied 

Genetics Comparison of the Peak Heights

FTA vs 903

11 year old bloodstains

0

1000

2000

3000

4000

0 1000 2000 3000 4000

FTA rfu

903 rfu

-20 C

20

37

x=y

11 year old bloodstains

0

1000

2000

3000

4000

0 1000 2000 3000 4000

FTA rfu

903 rfu

-20 C

20

37

x=y

Sample 1 Sample 2

Allelic dropout seen in some of the +37 C stored samples.

Those alleles were recovered with minimum length amplicons.
Applied 

Genetics 25 year old Bloodstain(1986)

Direct amp kit (903 paper)

Single 1.2 mm punch stored at room temperature

No Extraction

Data from Pete Vallone and Erica Butts
Applied 

Genetics Extracted DNA

• Factors:

–Liquid or Dried

• Storage Media

–Tube type (liquid)

–Storage Paper-Matrix (dried)

• Storage temperature

• Environmental excursions 

(planned or unplanned)
Applied 

Genetics

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 1 2 3 4 5

analysis time point 

[DNA] ng/μL

Each line represents an SRM 2391b component and the points along 

the lines (open circles) correspond to DNA concentrations obtained in 

this analysis. Several components (2, 8, 9,and 10) were approaching 

the original nominal DNA concentration of 1 ng/μL at the last time point 

of 4 days.

DNA sticking and releasing from tube walls

Original [DNA]

DNA stored in tubes 

for 5 years at -20 °C

then moved to 4 °C

Days at 4 °C
Applied 

Genetics

6 y DNA Extract Stability in Teflon tubes

19.0

20.0

21.0

22.0

23.0

24.0

25.0

Dilutions

Ct

-80 C

4 C

rm temp

6 year Extracted DNA Stability in PFA Tubes

Data from DNA extracts 

stored in PFA tubes at -80 °C, 

4 °C and room temperature 

for 6 years.

Each storage temperature 

had had 3 DNA 

concentrations neat, 1→5, 

1→10 dilutions.

Results are the qPCR Cts. 

Error bars representing 2 sd.

There is no difference as a result of temperature storage after 6 years.
Applied 

Genetics Study Design with Additive

• Stored DNA extracts with preservation additive. 

Four 96 well plates prepared

– 3 different concentrations

– 1 blank (no DNA)

• Control DNA without additive stored in the PFA 

containers at 4 C.
Applied 

Genetics

Biomatrica Shipped

5

10

15

20

25

30

35

40

45

50

55

6/19

7/4

7/19

8/3

8/18

9/2

9/1710/2

10/17

11/1

11/16

12/1

12/1612/31

1/15

Temperature C 

0

20

40

60

80

% Relative Humidity

“Shipped/Stressed” Temperature &

% Relative Humidity Profile, 208 days

Two plates with additive were 

“shipped” back and forth 

between MD and CA during 

the Summer of 2007.

After 6 cross country trips the 

plates were placed in a Car 

trunk for 14 days.

Two more cross country trips.

Followed by exposure to 

ambient attic temperatures 

for 56 days. 

Finally plates were placed at 

lab ambient conditions.

US

mail

Car

trunk

Attic

Lab

Max: 51.6 C, 73 % RH

Min: 5.3 °C, 15 % RH

Median: 22.1 °C, 40 % RH

Avg: 23.6 °C, 39 % RH

PFA Tube

added
Applied 

Genetics 208 Day Quantitation Results

[DNA]

ng/μL

Shipped

Stressed

Lab

ambient

Shipped 

PFA 

(147 days)

4 C

PFA

a 1 0.65 0.06 0.69 0.03 1.00 0.02 1.01 0.02

b 0.25 0.18 0.03 0.20 0.01 0.25 0.02 0.30 0.01

c 0.05 0.04 0.00 0.04 0.06 0.04 0.01 0.05 0.00

The decrease in the DNA concentration of the materials 

added to the plates with additive had been noted earlier, 

based on the genotyping results obtained the 

quantification data may be influenced by the color 

present in the additive.
Applied 

Genetics PFA Stored at 4 C

versus PFA Shipped

Blue symbols are 

from 4 C.

Red Symbols from 

harsh “ambient” 

conditions.

Teflon 4 C 208 days and Teflon "shipped 

"0.5 ng" amplified

Peak Heights

0

1000

2000

3000

4000

5000

6000

0 1000 2000 3000 4000 5000 6000

Lab stored

Shipped

B_a1

B_a2

x=y
Applied 

Genetics

So you want harsher conditions?

Or DNA extracts dried on paper?

OK
Applied 

Genetics X Ray screening of DNA Samples?

X Ray Package screening machines 

are found in more places than 

airport security lines these days.

It’s “rumored” that shipping 

companies such as UPS and FedEx 

randomly screen shipped packages.

Could shipping DNA /evidence to 

other laboratories be a problem? 

When we are conducting interlaboratory 

studies are the results bias due to 

shipping conditions?
Applied 

Genetics Study High Dose Irradiation 

(Sterilization)

• 5 MeV industrial x-ray beam

• Alanine dosimeter 

– (NIST reference class)

• Magazine Container

– Dose (91 ±4) kGy

– Max temp (57.5 ±1) °C

• Letter Mail Container

– Dose (87 ±4) kGy

– Max temp (62.5 ±1) °C Sample Packages were at 

these temperatures for 

20 to 30 minutes. 
Applied 

Genetics Experiment Parameters

• DNA extracts at 2 concentrations

 with and without stabilizer

• Stored in 3 different Tubes types

 with and without stabilizer 

 Stored as liquid or dried

• Stored as “Stains” on paper:

 FTA: 

 903: with and without stabilizer
Applied 

Genetics One set of Irradiation Study samples
Applied 

Genetics Treatments

• Control : Stored 4 °C

• Lab Ambient: 

Stayed in the lab until processed

• Shipped: 

Shipped UPS, returned UPS (?diagnostic x-rayed)

15 days in transit to and from (Washington State Patrol)

• X Rayed: 

Shipped UPS, X-rayed when received, returned UPS

7 days in transit (Evidence Control Unit FBI, VA)

• High-Dose Sterilization X Ray:

U.S. Postal Service Government Mail 
Applied 

Genetics

Flash Gel Results 

Post Treatment

Tube A

Tube B

Tube C

4 °C

Lab 

Ambient Shipped Low 

X Ray

High

X Ray
Applied 

Genetics Tube A Sample Results

95 % volume loss 100 % volume loss 95 % volume loss

The peak heights of allele 1 are plotted on the X-axis 

The peak heights of allele 2 are plotted on the Y-axis

Error bars span from the minimum to the maximum

The large symbols represent the average over all loci

Paper_Amb

Paper_Ship

Paper_Xray

Paper_Irad

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Tube A, Lab Ambient

Liquid

Dry

Dry-BM

Paper_Amb

Paper_Ship

Paper_Xray

Paper_Irad

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Tube A, Shipped

Liquid

Dry

Dry-BM

Paper

Paper

Paper

Paper

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Tube A, X-rayed

Liquid

Dry

Dry-BM
Applied 

Genetics Tube B Sample Results

14 % volume loss 33 % volume loss 15 % volume loss

The peak heights of allele 1 are plotted on the X-axis 

The peak heights of allele 2 are plotted on the Y-axis

Error bars span from the minimum to the maximum

The large symbols represent the average over all loci

Paper_Amb

Paper_Ship

Paper_Xray

Paper_Irad

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Tube B, Lab Ambient

Liquid

Dry

Dry-BM

Paper_Amb

Paper_Ship

Paper_Xray

Paper_Irad

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Tube B, Shipped

Liquid

Dry

Dry-BM

Pa

Pa

Pa

Pa

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Tube B, X-rayed

Liquid

Dry

Dry-BM
Applied 

Genetics Tube C Sample Results

0 % volume loss 1 % volume loss 0 % volume loss

The peak heights of allele 1 are plotted on the X-axis 

The peak heights of allele 2 are plotted on the Y-axis

Error bars span from the minimum to the maximum

The large symbols represent the average over all loci

Paper_Amb

Paper_Ship

Paper_Xray

Paper_Irad

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Tube C, Lab Ambient

Liquid

Dry

Dry-BM

Paper_Amb

Paper_Ship

Paper_Xray

Paper_Irad

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Tube C, Shipped

Liquid

Dry

Dry-BM

Pap

Pap

Pap

Pap

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Tube C, X-rayed

Liquid

Dry

Dry-BM
Applied 

Genetics High Irradiation Sample Results

100 % volume loss

The peak heights of allele 1 are plotted on the X-axis 

The peak heights of allele 2 are plotted on the Y-axis

Error bars span from the minimum to the maximum

The large symbols represent the average over all loci

No full profiles were obtained - only a few scattered alleles amplified.

17 % volume loss 19 % volume loss

Paper_Amb

Paper_Ship

Paper_Xray

Paper_Irad

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Tube A, High Irrad

Liquid

Dry

Dry-BM

Paper_Amb

Paper_Ship

Paper_Xray

Paper_Irad

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Tube B, High Irrad

Liquid

Dry

Dry-BM

Paper

Paper

Paper

Paper

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Tube C, High Irrad

Liquid

Dry

Dry-BM
Applied 

Genetics

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Paper, X-rayed

FTA

903

903-BM

Extracted DNA on paper

FTA: multiple allele loss

903: 1 allele loss

903BM: no loss

FTA: 30 μL of 2 ng/μL 

extracted DNA spotted

903: 30 μL of 2 ng/μL 

extracted DNA spotted

903-BM: 30 μL of 2 ng/μL 

extracted DNA spotted 

with stabilizer added

2 mm punches (≈1.6 ng) 

amplified in 25 μL 

reaction volume.

Error bars span from the 

minimum to the maximum

The large symbols 

represent the average 

over all loci

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Paper, Lab Ambient

FTA

903

903-BM

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Paper, Shipped

FTA

903

903-BM

FTA: multiple allele loss

903: 2 allele loss

903BM: no loss

FTA: multiple allele loss

903: no allele loss

903BM: no loss

0 1000 2000 3000 4000

0 1000 2000 3000 4000

Allele 2, RFU

Allele 1, RFU

Paper, High Irrad

FTA

903

903-BM

FTA: no alleles amplified

903: 1 allele amplified

903BM: 1 allele amplified
Applied 

Genetics Bottomline

• Tube packaging makes a difference

• Stabilizer doesn’t hurt, may help

• Getting a typing result from paper stain 

requires more DNA than you think

• Low dose X Ray doesn’t hurt

• High dose X Ray destroys
Applied 

Genetics

A partnership between the National Institute of 

Standards and Technology, Law Enforcement 

Standards Office and the National Institute of 

Justice, Office of Investigative and Forensic 

Sciences

Technical Working Group on 

Biological Evidence Preservation
Applied 

Genetics

The NIST/NIJ Technical Working Group on Biological 

Evidence Preservation (TWGBEP) is charged with: 

creating best practices and guidance to ensure 

the integrity, prevent the loss, and reduce the 

premature destruction of biological evidence 

after collection through post-conviction 

proceedings.

Technical Working Group on 

Biological Evidence Preservation
Applied 

Genetics Acknowledgments

Contact Info: 

margaret.kline@nist.gov

301-975-3134

NIST Team for This Work

Funding from the 

National Institute of 

Justice (NIJ) through 

NIST Office of Law 

Enforcement Standards

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.

Pete 

Vallone

Becky 

Hill

John 

Butler

Group Leader

Erica

Butts

Dave

Duewer
Applied 

Genetics Thank you for your attention!