Sample Lysis and DNA Separation in Single Tube Assemblies Abstract

Sample Lysis and DNA Separation in Single Tube Assemblies
for Accurate Forensic Profiling
Dr. Helge Schnerr – Biotype Diagnostic GmbH, Moritzburger Weg 67, 01109 Dresden, Germany
Abstract
The importance to improve quantity and quality of DNA isolated from forensic samples is without controversy. High quality extraction of
genomic DNA, as the first step of DNA profiling, is most critical for successful downstream Short Tandem Repeat (STR) analysis. Therefore,
disruption of cellular structures and separation of the soluble DNA fraction from cell debris needs to be optimized. Overall efficiency and yield
of the particle free DNA lysate will be improved by carrying out the lysis directly in a single tube assembly concomitantly allowing subsequent
quantitative lysate recovery.
Sampletype i-sep® DL
Sampletype i-sep® SQ
Unique to the DL version is the gradual DNA extraction required for
differential lysis of mixed specimens. Moreover, an immunological pretest for semen can be carried out using the identical sample. The single
tube assembly thus joins maximum comfort with most accurate
performance.
The SQ extraction systems mediates economic savings, reduces the
risk for cross contamination while improving the yield and quality of
DNA isolated from forensic samples such as swabs. It allows simple
and fast separation of substrate from lysate in an all-in-one system.
Thus, manual lysate and substrate transfer steps are eliminated, which
significantly rationalizes the work flow as exemplarily shown below:
Experiment: Differential lysis was performed under mild conditions to
separate the female DNA and harsher lysis conditions that break open
the spermatozoa as depicted below:
The DNA lysates were further purified and an autosomal STR profile of
both the victim and the perpetrator were generated using the particular
volumes of DNA-eluate.
Experiment: To demonstrate flexibility of the system in terms of lysis
conditions and required reagents a variety of lysis protocols were tested
(data not shown). Further, a rapid and inexpensive alternative to DNA
isolation for polymerase chain reaction (PCR) amplification from blood
was developed.
A volume of 36 µl 1 M Tris, pH 7.4 were added to the collection tube. A
filter column containing the sample (dried blood on textile 4 x 4 mm)
was inserted. A volume of 200 µl 0.1 N NaOH was added and incubated
on a shaker (550 rpm, 5 min). The assembly was centrifuged at 13.400
rpm for 1 min. The resulting lysate was mixed well and 5 µl were used
directly in the PCR.
6 µl
Conventional
Extraction
methode
17 µl
Sampletype i-sep® SQ
6 min DNA-ExpressIsolation
4 µl
Sampletype
i-sep® DL
1 µl
*Data provided by State Office of Criminal Investigation Saxony
Results: High quality yields of extracted DNA optimized down-stream
profiling. Thereby, simple handling allowed time savings and higher
throughput. Together, the DL-version has clear potential to reliably
improve crime solution rates and to speed up analysis of backlogged
crime samples.
*Data provided by State Office of Criminal Investigation Saxony
Results: High quality results, comparable to conventionally determined
results, were achieved using a 6-min-DNA-Express-Isolation. So,
Sampletype i-sep® SQ replaces present approaches requiring DNA
purification prior to PCR by combining incubation and separation with
high quality performance. The simple protocol is fast, easy to perform,
cost-effective, and consequently ideal for large sample numbers in the
routine. Furthermore, the Sampletype approach avoids anyway
switching to another tube. Thus eliminating the two-man rule during
sample preparation saving resources and minimizing the risk of cross
contamination and sample transposition events significantly.
Conclusion
Sampletype i-sep® extraction systems dramatically streamline the off-line lysis portion of the extraction method. Improvements of overall
efficiency, in particular maximizing the performance of the early steps of the extraction method has been demonstrated to achieve optimal
genotyping results. Ethylene oxide (EO) sterilization, according to the recommendations of the German Federal Criminal Police offices, has
been applied to guarantee DNA-free products.
In addition, Sampletype i-sep® DL can be rated as an improved methodology to overcome the often claimed difficulties in differential
extraction, thus, has the potential to work off backlogs of rape kits estimated at ~500.000 alone in the US1. Consecutive fractions of several
lysates are prepared by simply centrifugation; even allowing for an immunological pre-test using the identical sample.
References
1
Norris JV, Manning K, Linke SJ, Ferrance JP, Landers JP, Expedited, Chemically Enhanced Sperm Cell Recovery from Cotton Swabs for Rape Kit Analysis. J. Forensic Sci., 52, 800-805 (2007).
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