TY - JOUR
T1 - Development and validation of a novel multiplexed DNA analysis system, InnoTyper® 21
AU - Brown, Hiromi
AU - Thompson, Robyn
AU - Murphy, Gina
AU - Peters, Dixie
AU - La Rue, Bobby
AU - King, Jonathan
AU - Montgomery, Anne H.
AU - Carroll, Marion
AU - Baus, James
AU - Sinha, Sid
AU - Wendt, Frank R.
AU - Song, Bing
AU - Chakraborty, Ranajit
AU - Budowle, Bruce
AU - Sinha, Sudhir K.
N1 - Funding Information:
This material is based upon work supported by the National Science Foundation under SBIR Grant No. 1230352. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. The authors wish to express thanks to Dr. Bob Gary, Department of Microbiology, Tulane Health Science Center for DNA samples of non-human DNA used in this research and Dr. Michael Baird, DNA Diagnostics Center for providing DNA samples for database analysis. We express our thanks to Mr. Jonathan Tabak for constructive discussions and editing of this article.
Publisher Copyright:
© 2017 Elsevier B.V.
PY - 2017/7/1
Y1 - 2017/7/1
N2 - We report here a novel multiplexed DNA analysis system consisting of 20 Alu markers and Amelogenin for analysis of highly degraded forensic biological samples. The key to the success of the system in obtaining results from degraded samples is the primer design yielding small amplicon size (60–125 bp) for all 20 markers. The markers included in the InnoTyper® 21 system are bi-allelic, having two possible allelic states (insertion or null) and thus termed INNULs. The markers are short interspersed nuclear elements (SINEs), a category of retrotransposable elements (REs) which are non-coding genomic DNA repeat sequences, or “mobile insertion elements,” comprising approximately 40% of the human genome. Alu elements are primate specific SINEs that have reached a copy number in excess of one million in the human genome, which makes these markers highly sensitive and desirable for forensic samples with extremely degraded DNA. Until now however, due to the inherent size difference associated with insertion and no insertion alleles, the use of Alu REs has not been practical for forensic applications. The novel primer design described herein has allowed the development of a multiplexed Alu system yielding fragment sizes amenable to degraded DNA samples, as frequently encountered in missing persons cases or forensic samples such as hair shafts. Although use of Alus in human identity has been studied using single marker amplification and reported before, we report for the first time development and validation of a system with multiplexed RE markers. Studies performed include PCR optimization, species specificity, sensitivity, degradation and inhibition, precision and accuracy, nonprobative samples, mixture, and population database studies. A population study using 592 samples including five populations was performed using InnoTyper 21. The data indicated the random match probability for the combination of these 20 Alu markers was greater than 1 in 3.8 million for the populations studied, indicating the greater statistical power of these autosomal nuclear DNA markers over haplotype systems typically used in such degraded samples. Results demonstrate the system is successful in obtaining results from highly degraded DNA. A sensitivity study performed demonstrated at least 95% recovery of alleles from as low as 50 pg of total input DNA, and partial profiles from as low as 25 pg. This study has demonstrated that the bi-allelic INNULs in the InnoTyper 21 system provide a sensitivity of detection and a power of discrimination that makes them useful for human identification of extremely degraded samples.
AB - We report here a novel multiplexed DNA analysis system consisting of 20 Alu markers and Amelogenin for analysis of highly degraded forensic biological samples. The key to the success of the system in obtaining results from degraded samples is the primer design yielding small amplicon size (60–125 bp) for all 20 markers. The markers included in the InnoTyper® 21 system are bi-allelic, having two possible allelic states (insertion or null) and thus termed INNULs. The markers are short interspersed nuclear elements (SINEs), a category of retrotransposable elements (REs) which are non-coding genomic DNA repeat sequences, or “mobile insertion elements,” comprising approximately 40% of the human genome. Alu elements are primate specific SINEs that have reached a copy number in excess of one million in the human genome, which makes these markers highly sensitive and desirable for forensic samples with extremely degraded DNA. Until now however, due to the inherent size difference associated with insertion and no insertion alleles, the use of Alu REs has not been practical for forensic applications. The novel primer design described herein has allowed the development of a multiplexed Alu system yielding fragment sizes amenable to degraded DNA samples, as frequently encountered in missing persons cases or forensic samples such as hair shafts. Although use of Alus in human identity has been studied using single marker amplification and reported before, we report for the first time development and validation of a system with multiplexed RE markers. Studies performed include PCR optimization, species specificity, sensitivity, degradation and inhibition, precision and accuracy, nonprobative samples, mixture, and population database studies. A population study using 592 samples including five populations was performed using InnoTyper 21. The data indicated the random match probability for the combination of these 20 Alu markers was greater than 1 in 3.8 million for the populations studied, indicating the greater statistical power of these autosomal nuclear DNA markers over haplotype systems typically used in such degraded samples. Results demonstrate the system is successful in obtaining results from highly degraded DNA. A sensitivity study performed demonstrated at least 95% recovery of alleles from as low as 50 pg of total input DNA, and partial profiles from as low as 25 pg. This study has demonstrated that the bi-allelic INNULs in the InnoTyper 21 system provide a sensitivity of detection and a power of discrimination that makes them useful for human identification of extremely degraded samples.
KW - Alu
KW - Degraded DNA
KW - InnoTyper 21
KW - Novel forensic markers
UR - http://www.scopus.com/inward/record.url?scp=85017171941&partnerID=8YFLogxK
U2 - 10.1016/j.fsigen.2017.03.017
DO - 10.1016/j.fsigen.2017.03.017
M3 - Article
C2 - 28391141
AN - SCOPUS:85017171941
SN - 1872-4973
VL - 29
SP - 80
EP - 99
JO - Forensic Science International: Genetics
JF - Forensic Science International: Genetics
ER -