Development and validation of a novel multiplexed DNA analysis system, InnoTyper® 21

Hiromi Brown, Robyn Thompson, Gina Murphy, Dixie Peters, Bobby La Rue, Jonathan King, Anne H. Montgomery, Marion Carroll, James Baus, Sid Sinha, Frank R. Wendt, Bing Song, Ranajit Chakraborty, Bruce Budowle, Sudhir K. Sinha

Research output: Contribution to journalArticleResearchpeer-review

8 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)80-99
Number of pages20
JournalForensic Science International: Genetics
Volume29
DOIs
StatePublished - 1 Jul 2017

Fingerprint

Alu Elements
DNA
Human Genome
Population
Alleles
Amelogenin
Species Specificity
Forensic Anthropology
Genetic Markers
Hair
Haplotypes
Primates
Databases
Polymerase Chain Reaction

Keywords

  • Alu
  • Degraded DNA
  • InnoTyper 21
  • Novel forensic markers

Cite this

Brown, Hiromi ; Thompson, Robyn ; Murphy, Gina ; Peters, Dixie ; La Rue, Bobby ; King, Jonathan ; Montgomery, Anne H. ; Carroll, Marion ; Baus, James ; Sinha, Sid ; Wendt, Frank R. ; Song, Bing ; Chakraborty, Ranajit ; Budowle, Bruce ; Sinha, Sudhir K. / Development and validation of a novel multiplexed DNA analysis system, InnoTyper® 21. In: Forensic Science International: Genetics. 2017 ; Vol. 29. pp. 80-99.
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title = "Development and validation of a novel multiplexed DNA analysis system, InnoTyper{\circledR} 21",
abstract = "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{\circledR} 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.",
keywords = "Alu, Degraded DNA, InnoTyper 21, Novel forensic markers",
author = "Hiromi Brown and Robyn Thompson and Gina Murphy and Dixie Peters and {La Rue}, Bobby and Jonathan King and Montgomery, {Anne H.} and Marion Carroll and James Baus and Sid Sinha and Wendt, {Frank R.} and Bing Song and Ranajit Chakraborty and Bruce Budowle and Sinha, {Sudhir K.}",
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Brown, H, Thompson, R, Murphy, G, Peters, D, La Rue, B, King, J, Montgomery, AH, Carroll, M, Baus, J, Sinha, S, Wendt, FR, Song, B, Chakraborty, R, Budowle, B & Sinha, SK 2017, 'Development and validation of a novel multiplexed DNA analysis system, InnoTyper® 21', Forensic Science International: Genetics, vol. 29, pp. 80-99. https://doi.org/10.1016/j.fsigen.2017.03.017

Development and validation of a novel multiplexed DNA analysis system, InnoTyper® 21. / Brown, Hiromi; Thompson, Robyn; Murphy, Gina; Peters, Dixie; La Rue, Bobby; King, Jonathan; Montgomery, Anne H.; Carroll, Marion; Baus, James; Sinha, Sid; Wendt, Frank R.; Song, Bing; Chakraborty, Ranajit; Budowle, Bruce; Sinha, Sudhir K.

In: Forensic Science International: Genetics, Vol. 29, 01.07.2017, p. 80-99.

Research output: Contribution to journalArticleResearchpeer-review

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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.

PY - 2017/7/1

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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.

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