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DNA Analysis of PCR-Inhibitory Forensic Samples

Hedman, Johannes LU (2011)
Abstract (Swedish)
Popular Abstract in Swedish

I Sverige analyseras omkring 40 000 DNA-prover från brottsplatser varje år. DNA-profilerna jämförs mot DNA-profiler från misstänkta personer och kan på så vis användas för att knyta en person till ett brott. Alla typer av kroppsvävnader kan analyseras, såsom blod, saliv och hudceller. Vanliga bevismaterial är cigarettfimpar, flaskor, burkar och kläder. De material som cellerna sitter på kan störa DNA-analysen, PCR, och därigenom försämra DNA-profilens kvalitet.



I november 2010 modifierades DNA-analysen av brottsplatsprover vid Statens

kriminaltekniska Laboratorium. Det enzym, DNA-polymeras, som dittills varit

standard i Sverige och i resten av världen ersattes... (More)
Popular Abstract in Swedish

I Sverige analyseras omkring 40 000 DNA-prover från brottsplatser varje år. DNA-profilerna jämförs mot DNA-profiler från misstänkta personer och kan på så vis användas för att knyta en person till ett brott. Alla typer av kroppsvävnader kan analyseras, såsom blod, saliv och hudceller. Vanliga bevismaterial är cigarettfimpar, flaskor, burkar och kläder. De material som cellerna sitter på kan störa DNA-analysen, PCR, och därigenom försämra DNA-profilens kvalitet.



I november 2010 modifierades DNA-analysen av brottsplatsprover vid Statens

kriminaltekniska Laboratorium. Det enzym, DNA-polymeras, som dittills varit

standard i Sverige och i resten av världen ersattes av en polymerasblandning som jag designat och prövat ut i detta doktorandprojekt. Den nya metoden gav kompletta DNA-profiler från ett ökat antal smutsiga prover med lite DNA. För saliv höjdes andelen från 38% till 87%, och för blod från 69% till 94%.



I inledande studier visade jag att de två polymeraserna som ingår i blandningen är mer robusta mot smuts jämfört med standardanalysen. De uppvisade också skillnader sinsemellan, exempelvis fungerade det ena bättre för snusprover medan det andra fungerade bättre för tuggummi och cigarettfimpar. Blandningen blev ett sätt att utnyttja dessa komplementära egenskaper till att skapa en mer generell metod. Förutom komplementaritet så uppvisade de två enzymerna synergi: de förstärkte varandras goda egenskaper.



Resultatet från en kriminalteknisk DNA-analys är ett komplext diagram, där toppar representerar DNA-profilen. Topparnas höjd och balans visar hur väl analysen fungerat. För att kunna jämföra prestandan mellan olika analysmetoder behöver man kvantifiera DNA-profilens kvalitet. I detta syfte utvecklade jag en matematisk modell som omvandlar analysdiagram till enskilda kvalitetsvärden. Modellen användes sedan för att bevisa synergin mellan de två DNA-polymeraserna i blandningen.



Saliv är vanligt förekommande på brottsplatser och kan säkras från exempelvis flaskor och burkar. Eftersom salivspår ofta är osynliga för blotta ögat ger många prover som förväntas innehålla saliv negativa DNA-analysresultat. För att minska andelen negativa prover utvecklade jag en enkel metod för att testa provtagningstops för närvaro av saliv. Metoden gav utslag ner till 0.5 μL torkad saliv, och kan användas som urvalsverktyg i exempelvis utredningar av inbrott. Testet kan utföras direkt på brottsplats utan laboratorieutrustning. (Less)
Abstract
DNA evidence, linking perpetrators to crime scenes, is central to many legal

proceedings. However, crime scene samples often contain extraneous substances that may interfere with the PCR-based forensic analysis, resulting in partial or negative DNA profiles. Extensive DNA purification may remove inhibitors, but involves the risk of DNA loss. In this work, pre-PCR processing was applied to improve the success rate of forensic DNA analysis of “dirty” samples without interfering with the composition of the samples.



An experimental model system was developed to screen for inhibitor-tolerant DNA polymerase-buffer systems. The best-performing polymerases, Bio-X-Act Short, ExTaq HS and PicoMaxx HF, were applied in STR... (More)
DNA evidence, linking perpetrators to crime scenes, is central to many legal

proceedings. However, crime scene samples often contain extraneous substances that may interfere with the PCR-based forensic analysis, resulting in partial or negative DNA profiles. Extensive DNA purification may remove inhibitors, but involves the risk of DNA loss. In this work, pre-PCR processing was applied to improve the success rate of forensic DNA analysis of “dirty” samples without interfering with the composition of the samples.



An experimental model system was developed to screen for inhibitor-tolerant DNA polymerase-buffer systems. The best-performing polymerases, Bio-X-Act Short, ExTaq HS and PicoMaxx HF, were applied in STR DNA analysis of PCRinhibitory crime scenes samples, i.e. samples that failed to produce complete DNA profiles in routine casework despite containing acceptable levels of DNA. A ranking index, called the forensic DNA profile index (FI), was developed to quantitatively describe DNA profile quality. The application of analysis of variance (ANOVA) to FI values confirmed that the three alternative polymerases significantly improved DNA profile quality for 20 of 32 problematic samples, compared with the standard polymerase AmpliTaq Gold. ExTaq HS and PicoMaxx HF showed complementary inhibitor-relieving properties. A blend of the two polymerases exhibited tolerance to a broader range of extraneous compounds, improving DNA profile quality in 34 of 42 PCR-inhibitory forensic samples. When used separately, ExTaq HS and PicoMaxx HF improved the results of analysis for 26 and 23 samples, respectively. Apart from their complementarity, synergy between the polymerases was mathematically proven by calculating the geometric mean values of FI and applying ANOVA.



In November, 2010, the customised DNA polymerase blend was introduced in

routine casework at the Swedish National Laboratory of Forensic Science,

increasing the proportion of complete DNA profiles generated from impure

samples from 38% to 87% for saliva, and from 69% to 94% for blood.



Many presumed saliva crime scene stains give negative DNA results if presumptive testing is not performed. In this work, amylase activity testing was evaluated as a tool for saliva screening. No direct correlation was found between amylase activity and the DNA content of saliva. However, the sensitivity of the developed swab screening procedure (positive results for 0.5 μL of dried saliva) makes it applicable in cases where the number of DNA analyses is limited due to cost. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Dr Butler, John M., Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA
organization
publishing date
type
Thesis
publication status
published
subject
keywords
amylase, statistical modelling, principal components analysis, PCR inhibitors, PCR inhibition, forensic DNA analysis, DNA polymerase blend, DNA polymerase, crime scene samples
pages
157 pages
defense location
Lecture hall B, Kemicentrum, Getingevägen 60, Lund University, Faculty of Engineering
defense date
2011-11-25 10:30
ISBN
978-91-7422-283-8
language
English
LU publication?
yes
id
20814676-a8e2-4cfc-85ed-45fecfef6e97 (old id 2198638)
date added to LUP
2011-10-27 12:43:15
date last changed
2016-09-19 08:45:15
@misc{20814676-a8e2-4cfc-85ed-45fecfef6e97,
  abstract     = {DNA evidence, linking perpetrators to crime scenes, is central to many legal<br/><br>
proceedings. However, crime scene samples often contain extraneous substances that may interfere with the PCR-based forensic analysis, resulting in partial or negative DNA profiles. Extensive DNA purification may remove inhibitors, but involves the risk of DNA loss. In this work, pre-PCR processing was applied to improve the success rate of forensic DNA analysis of “dirty” samples without interfering with the composition of the samples.<br/><br>
<br/><br>
An experimental model system was developed to screen for inhibitor-tolerant DNA polymerase-buffer systems. The best-performing polymerases, Bio-X-Act Short, ExTaq HS and PicoMaxx HF, were applied in STR DNA analysis of PCRinhibitory crime scenes samples, i.e. samples that failed to produce complete DNA profiles in routine casework despite containing acceptable levels of DNA. A ranking index, called the forensic DNA profile index (FI), was developed to quantitatively describe DNA profile quality. The application of analysis of variance (ANOVA) to FI values confirmed that the three alternative polymerases significantly improved DNA profile quality for 20 of 32 problematic samples, compared with the standard polymerase AmpliTaq Gold. ExTaq HS and PicoMaxx HF showed complementary inhibitor-relieving properties. A blend of the two polymerases exhibited tolerance to a broader range of extraneous compounds, improving DNA profile quality in 34 of 42 PCR-inhibitory forensic samples. When used separately, ExTaq HS and PicoMaxx HF improved the results of analysis for 26 and 23 samples, respectively. Apart from their complementarity, synergy between the polymerases was mathematically proven by calculating the geometric mean values of FI and applying ANOVA.<br/><br>
<br/><br>
In November, 2010, the customised DNA polymerase blend was introduced in<br/><br>
routine casework at the Swedish National Laboratory of Forensic Science,<br/><br>
increasing the proportion of complete DNA profiles generated from impure<br/><br>
samples from 38% to 87% for saliva, and from 69% to 94% for blood.<br/><br>
<br/><br>
Many presumed saliva crime scene stains give negative DNA results if presumptive testing is not performed. In this work, amylase activity testing was evaluated as a tool for saliva screening. No direct correlation was found between amylase activity and the DNA content of saliva. However, the sensitivity of the developed swab screening procedure (positive results for 0.5 μL of dried saliva) makes it applicable in cases where the number of DNA analyses is limited due to cost.},
  author       = {Hedman, Johannes},
  isbn         = {978-91-7422-283-8},
  keyword      = {amylase,statistical modelling,principal components analysis,PCR inhibitors,PCR inhibition,forensic DNA analysis,DNA polymerase blend,DNA polymerase,crime scene samples},
  language     = {eng},
  pages        = {157},
  title        = {DNA Analysis of PCR-Inhibitory Forensic Samples},
  year         = {2011},
}