Direct PCR amplification of DNA from human bloodstains, saliva, and touch samples collected with microFLOQ® swabs.

Previous studies have shown that nylon flocked swabs outperform traditional fiber swabs in DNA recovery due to their innovative design and lack of internal absorbent core to entrap cellular materials. The microFLOQ® Direct swab, a miniaturized version of the 4N6 FLOQSwab®, has a small swab head that is treated with a lysing agent which allows for direct amplification and DNA profiling from sample collection to final result in less than two hours. Additionally, the microFLOQ® system subsamples only a minute portion of a stain and preserves the vast majority of the sample for subsequent testing or re-analysis, if desired. The efficacy of direct amplification of DNA from dilute bloodstains, saliva stains, and touch samples was evaluated using microFLOQ® Direct swabs and the GlobalFiler™ Express system. Comparisons were made to traditional methods to assess the robustness of this alternate workflow. Controlled studies with 1:19 and 1:99 dilutions of bloodstains and saliva stains consistently yielded higher STR peak heights than standard methods with 1ng input DNA from the same samples. Touch samples from common items yielded single source and mixed profiles that were consistent with primary users of the objects. With this novel methodology/workflow, no sample loss occurs and therefore more template DNA is available during amplification. This approach may have important implications for analysis of low quantity and/or degraded samples that plague forensic casework.

[1]  Duncan A. Taylor,et al.  Direct PCR Improves the Recovery of DNA from Various Substrates , 2015, Journal of forensic sciences.

[2]  Stefan Lorkowski,et al.  Characterization of the synthetic compatible solute homoectoine as a potent PCR enhancer. , 2004, Biochemical and biophysical research communications.

[3]  W. Al-Soud,et al.  Purification and Characterization of PCR-Inhibitory Components in Blood Cells , 2001, Journal of Clinical Microbiology.

[4]  Cristina E. Valdiosera,et al.  Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments , 2013, Proceedings of the National Academy of Sciences.

[5]  J. I. Bell,et al.  Effect of heparin on polymerase chain reaction , 1994, The Lancet.

[6]  R. Findlay,et al.  Convenient determination of DNA extraction efficiency using an external DNA recovery standard and quantitative-competitive PCR. , 2004, Journal of microbiological methods.

[7]  R. Ravazzolo,et al.  Betaine, dimethyl sulfoxide, and 7-deaza-dGTP, a powerful mixture for amplification of GC-rich DNA sequences. , 2006, The Journal of molecular diagnostics : JMD.

[8]  J. Carpten,et al.  Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. , 1996, BioTechniques.

[9]  Young-Han Song,et al.  A novel buffer system, AnyDirect, can improve polymerase chain reaction from whole blood without DNA isolation. , 2007, Clinica chimica acta; international journal of clinical chemistry.

[10]  W. Al-Soud,et al.  Identification and Characterization of Immunoglobulin G in Blood as a Major Inhibitor of Diagnostic PCR , 2000, Journal of Clinical Microbiology.

[11]  C. Schutt,et al.  The enhancement of PCR amplification by low molecular weight amides. , 2001, Nucleic acids research.

[12]  B. Kemp,et al.  How Much DNA is Lost? Measuring DNA Loss of Short-Tandem-Repeat Length Fragments Targeted by the PowerPlex 16® System Using the Qiagen MinElute Purification Kit , 2015, Human biology.

[13]  J. Clark,et al.  Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. , 1988, Nucleic acids research.

[14]  B. Kokshoorn,et al.  Response to Grisedale and Van Daal: comparison of STR profiling from low template DNA extracts with and without the consensus profiling method , 2013, Investigative Genetics.

[15]  C. Schutt,et al.  Novel sulfoxides facilitate GC-rich template amplification. , 2002, BioTechniques.

[16]  A. Garvin,et al.  Purifying and Concentrating Genomic DNA from Mock Forensic Samples Using Millipore Amicon Filters , 2013, Journal of forensic sciences.

[17]  B. Kemp,et al.  One of the key characteristics of ancient DNA, low copy number, may be a product of its extraction , 2014 .

[18]  R. J. Mitchell,et al.  Swabs as DNA Collection Devices for Sampling Different Biological Materials from Different Substrates , 2014, Journal of forensic sciences.

[19]  R. Dirnhofer,et al.  Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains. , 1991, Journal of forensic sciences.

[20]  Guohua Zhou,et al.  Direct polymerase chain reaction (PCR) from human whole blood and filter-paper-dried blood by using a PCR buffer with a higher pH. , 2008, Analytical biochemistry.

[21]  P. Singh,et al.  A novel method for whole blood PCR without pretreatment. , 2012, Gene.

[22]  D. Gorenstein,et al.  The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers. , 2005, Journal of biochemical and biophysical methods.

[23]  W Henke,et al.  Betaine improves the PCR amplification of GC-rich DNA sequences. , 1997, Nucleic acids research.

[24]  A. Barbaroa,et al.  DNA profiling by different extraction methods , 2004 .

[25]  Kathryn E. Dagnall,et al.  A Comparison of DNA Collection and Retrieval from Two Swab Types (Cotton and Nylon Flocked Swab) when Processed Using Three QIAGEN Extraction Methods , 2012, Journal of forensic sciences.

[26]  Duncan A. Taylor,et al.  Genetic profiling from challenging samples: Direct PCR of touch DNA , 2013 .

[27]  A Walton,et al.  Automated DNA profiling employing multiplex amplification of short tandem repeat loci. , 1993, PCR methods and applications.

[28]  M. Kermekchiev,et al.  Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq. , 2010, The Journal of molecular diagnostics : JMD.

[29]  Johannes Hedman,et al.  Purification of crime scene DNA extracts using centrifugal filter devices , 2013, Investigative Genetics.

[30]  H. Lehrach,et al.  An efficient and economic enhancer mix for PCR. , 2006, Biochemical and biophysical research communications.

[31]  David R Foran,et al.  Assessment and mitigation of DNA loss utilizing centrifugal filtration devices. , 2014, Forensic science international. Genetics.

[32]  Chien-Wei Chang,et al.  Direct amplification of STRs from blood or buccal cell samples , 2009 .

[33]  A. Akane,et al.  Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. , 1994, Journal of forensic sciences.

[34]  Joseph B. Rayman,et al.  Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: implications for PCR-based genotyping and cloning. , 1996, BioTechniques.