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Average Signal Increase across all Amplification Kits

0

5

10

15

20

25

30

35

Identifiler Cofiler ProfilerPlus

PowerPlex 16

Minifiler Y Filer PowerPlex Y

S5

Amplification Kits

Ave

rage

Sig

nal I

ncre

ase

gn526510.0gn52130.0

Identifiler 0.0625ng

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

13,1

4

28,3

0

11,1

2

11,1

2

15,1

7 7

9,12

9,11

23,2

4

14,1

6.2

15,2

0 8

13,1

7

X,Y 11

21,2

5

D8 D21 D7 CSF D3 THO1 D13 D16 D2 D19 vWATPOXD18 Amel D5 FGA

Loci/ Profile

Rfu

s

Before Clean upAfter Clean up

Power Plex 16 0.0625ng

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

15,1

7 7

28,3

0

13,1

7

7,13 11

9,12

11,1

2

9,11

11,1

2

11,1

2

X,Y

15,2

0

13,1

4 8

21,2

5

D3 THO1 D21 D18 PentaE

D5 D13 D7 D16 CSF PentaD

Amel vWA D8 TPOX FGA

Loci/ Profile

Rfu

s Before Clean upAfter Clean up

-A after clean up procedure is now 50% and not filtered out

-A before clean up 38%

Dye Blob present before cleanup procedure

Dye blobs did not increase in proportion to true peaks. In some instances the dye blobs were reduced in size and were even removed after clean up.

Dye blob after post PCR clean up increased in height 2.2 times while true allele increased in height 10 times

Drop out of data occurs at the same

Elevated stutter before clean up

Drop out of data from Loci DYS19 and

DYS392 before clean up procedure

Drop out of data still occurs at the

same loci after clean up procedure

Elevated stutter still present after clean up

Any issues with the amplification of low level DNA samples such as drop in, high stutter, preferential amplification, and differential amplification were still present after clean up.

After clean up 15,17, profile only raised above 75RFUs.

D3S1358 before clean up no peaks discerned from baseline. At this locus 15,17 is the profile of the sample

In some instances peaks that were not originally discernable from the baseline were clearly identified after the purification process.

Heterozygosity 81% Heterozygosity 47% Heterozygosity 9 7%

Heterozygosity 48% Heterozygosity 80% Heterozygosity 93%

In some instances there were fluctuations in heterozygosity when comparing samples pre and post clean up. There was no discernable trend or cause for these fluctuations.

1 National Forensic Science Technology Center, 7881 114th Ave North, Largo, FL 33773

Abstract:This study was designed to evaluate the similarities and differences in capillary electrophoresis signal detection when using the QIAGEN MinElute® PCR Purification Kit on amplified DNA obtained from commonly used short tandem repeat (STR) commercial amplification kits.

The following commercially available STR amplification kits used in this study were:

Applied Biosystems’ AmpflSTR®: Promega’s:

• Profiler Plus® ID kit •PowerPlex® 16 system • Cofiler® kit •PowerPlex® Y system• Identifiler® kit •PowerPlex® S5 system• MiniFilerTM kit • Yfiler® kit

Serial dilutions prepared from DNA extracts ranging from 1.0 ng to 0.0078 ng were evaluated on each of the amplification kits. Concentrations at which data fell below 75 RFUs were run through the clean up columns.

Introduction:This study was conducted to demonstrate the benefits of includ-ing the QIAGEN MinElute® PCR Purification Kit into the analysis scheme on forensic samples containing low quantities of DNA.

The QIAGEN MinElute® PCR Purification Kit uses a silica membrane to bind DNA fragments ranging in size from 70 bp to 4kb. While the DNA is bound to the membrane, impurities such as unwanted primers, salts, enzymes, unincorporated nucle-otides , dyes, oils, and detergents flow through the column. This causes an increase in the signal from the amplified DNA which in turn increases the peak height of the resulting data allowing previously low level samples to reach to a callable level. Removal of these impurities ensures that more DNA is injected during the electrokinetic injection on the instrumentation, thus increasing the fluorescent signal intensity.

Materials:• Standards from 2 male donors• Phenol:chloroform:Isoamyl alcohol (25:24:1)• TE Buffer, DTT and Proteinase K (10ng/ul)• Applied Biosystems Human DNA Quantifiler® Kit• Applied Biosystems’ AmpflSTR® Profiler Plus® kit, Cofiler® kit, Identifiler® kit, MiniFiler™ kit, and the Yfiler® • Promega’s PowerPlex® 16 system, PowerPlex® Y system,

and the PowerPlex® S5 system• Running Buffer, 10X • 16 capillary array, 36cm• POP-4TM polymer for 3130xl• Matrix standards• Internal Lane Size Standards • Hi-DiTM Formamide• 96-Well GeneAmp® PCR System 9700• 7500 Real-Time PCR System• 3130xl Genetic Analyzer• QIAGEN MinElute® PCR Purification Kit.

Method:• Two separate known human male DNA standards were prepared utilizing a standard organic extraction method in conjunction with the Millipore Microcon® 100 centrifugal filter device.

• Serial dilutions were prepared from DNA extracts at the following concentrations: 1.0, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, and 0.0078 ng.

• The samples were quantitated using the Applied Biosystems Quantifiler®Human Quantification Kit on an Applied Biosystems 7500 Real-Time PCR System. The results were normalized with NIST quantitation standards.

• Samples were amplified on an Applied Biosystems GeneAmp® PCR 9700 thermal cycler following manufacturer’s specifications.

• The samples were then separated and detected using an Applied Biosystems 3130xl Genetic Analyzer using manufacture’s recommendations:

— Applied Biosystems kits: 3kv, 10 sec injections, 8.7 µl Formamide, 0.3 µl GS 500, 1 µl sample.

— Promega kits: 3kv, 10 sec injections, 1200 sec run time, 9.5 µl Formamide, 0.5 µl ILS 600, 1 µl sample

• Data were analyzed using GeneMapper® ID Software v3.2 using a threshold of 75 RFUs.

• Amplified samples from these dilutions which had data falling below a 75 RFU threshold were purified using the QIAGEN MinElute® PCR Purification Kit. A total of 4 washes were performed manually on each sample.

• After the post PCR clean up procedure the samples were then run on the Applied Biosystems 3130xl Genetic Analyzer under the same conditions. All of the purified amplification product, which ranged between 9 and 10 µl, was consumed

Benefit to the Forensic Community: Crime laboratories have seen an increase in the submission of requests for analysis on evidentiary items with low quantities of DNA. This study demonstrated that the QIAGEN MinElute® PCR Purification Kit consistently increased the fluorescent signal with the eight evaluated commercial STR amplification kits. This purification kit can be integrated into the laboratory process with little effort for method validation and at minimal cost. Integration of the QIAGEN MinElute® PCR Purification Kit into the DNA analysis procedure is a simple, cost effective method that can be easily implemented by a crime laboratory to increase the overall sensitivity of their DNA analysis methods.

References:Smith, PJ, Ballantyne J. “Simplified Low-Copy-Number DNA Analysis by Post-PCR Purification.” Journal of Forensic Science, 52(4). (2007):820-829

MinElute Handbook [Internet]. [updated 2008 Mar]. QIAGEN. [cited 2008 August 14]. Available from:

http://www1.QIAGEN.com/Products/DNACleanup/GelPcrSiCleanupSystems/MinElutePCRPurificationKit.aspx#Tabs=t2

The most current user and technical manuals for each kit were referenced in this study. Manuals procured from www.appliedbiosystems.com (published 1998-2006) and www.promega.com/tbs/ (published 2008.)

Study Contact and Author Affiliations:1National Forensic Science Technology Center7881 114th Avenue NLargo, FL 33773727-549-6067

www.nfstc.org

Results and Discussion:Conclusions:This method of post PCR clean up consistently increased the signal of samples which were previously at levels too low to be characterized. Laboratories should perform appropriate validation studies to establish interpretation guidelines to account for the issues which occur with amplification of low level DNA samples.

After Post PCR clean up there was a signal increase from an original peak height of 50 RFUs to a minimum of 300 RFUs in every kit tested.

Minus A (-A) present prior to clean up increased after clean up in a greater proportion than that of the allele. If no -A was present initially, then there was no -A after clean up.

The Benefit of Using the QIAGEN MinElute® PCR Purification Kit for Post PCR Cleanup on Low Level DNA Samples

Study funded through Cooperative Agreement 2008-MU-MU-K003 with the National Institute of Justice, the research, development and evaluation agency of the U.S. Department of Justice. The NFSTC manages the Forensic Technologies Center of Excellence (FTCoE) through this agreement.

O’Brien, Robert I, BS1*; Sutherland, Carrie B, BS1; Figarelli, Debra A, BS1; Ring, Joan G, MS 1, and Grates, Kirk M, BA1