Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.
Ashley Manning Hart                 University at Albany          In partial fulfillment of theMaster of Science in Forens...
 Extraction Quantitation Amplification STR DNA Analysis & Interpretation Report writing Review Testimony
 STR DNA analysis is very sensitive Inhibition of samples by either substrate or  sample itself Wide range of amounts a...
   Crime scene samples can be “dirty” and contaminated    with non-human DNA   Microbial, plant, and/or animal DNA co-ex...
 Ease of interpretation – especially with DNA  mixtures Comparison  purposes of evidence samples to  reference samples ...
 Known    samples    Reference samples    Offender samples Questioned    samples    Single source    Mixtures: male/...
   Quantifiler Y Human Male DNA Quantification Kit    detects male DNA   Most valuable when male:female DNA ratio low:  ...
 UV   Spectrometry Gel-based   Methods Real-time   PCR Methods
 Based on stacking of nitrogenous bases Stacking limits the amount of UV light  absorbed Disadvantages:    Cannot dist...
 Gelsor blotting membranes utilized DNA specific dyes as reporter molecules    Species non-specific Advantages:    Br...
 Applied Biosystems method – chemiluminescence  or chromogenic Slot blot on a nylon membrane HUMAN AND HIGHER PRIMATE ...
 Commonly    used: Alu probes – more areas  targeted as compared to slot blot methods Fluorescent probes Very sensitive...
 Advantages    Less time-consuming than other methods    Less hands-on time  ability to be automated Disadvantages  ...
 Targetshaploid 64-base length segment, Yp11.3 – the non-translated portion of the sex-determining region of the Y-chromo...
-Two TaqMan probes target: untranslated region of SRY gene andIPC- Reporter dye (FAM for DNA of interest, VIC for IPC) on ...
-PCR temperature increased to 72ºC  P moves 5’ – 3’ and extendsforward and reverse primers- Nucleotides (from Reaction Mi...
 PRIMER MIX: Human Target Specific Assay    •   FAM labeled probe (blue reporter dye) – targets        Yp11.3   Interna...
   Human male DNA, 200ng/ L – supplied with kit   Standard curve: 50ng/ L to 23pg/ L   Comparison of Q sample fluoresce...
 96-well optical plate 7500 Real-Time PCR instrument with  Sequence Detection Software 3 phases of PCR: geometric (expo...
 Fluorescenceemitted by release of reporter dye in sample  detected by camera Fluorescencethreshold set by Applied Bios...
 Varies depending on amount of DNA in  sample: well with more DNA will have a  lower CT because it takes fewer cycles for...
 Validation:  to recognize, establish, or  illustrate the worthiness or legitimacy of Validation of new techniques is es...
 FBI   Quality Assurance Standard 8.1.3     8.1.3.1 – Testing of known and non-probative      evidence samples & Documen...
   DNA extracts from simulated evidence   Quantities and CT values compared   Comparisons to the Amelogenin locus resul...
   QuantiBlot performed by other analysts   Quantifiler and Quantifiler Y combined on one assay
Sample           Percentage Male DNA              Electropherogram   Quantifiler YJRK 5.5 NSF         5.57             7.1...
CT Sample                    CT IPC                          CT Sample                  CT IPCSample Type Quantifiler     ...
 Using Quantifiler Y Kit, many slopes fell  below AB’s recommended range (-3.0 to -3.6) Erratic slope patterns using lot...
-Log of concentration plotted against CT values- Slope outside of acceptable, AB-validated range  overestimate ofDNA conc...
 Issues not resolved so emphasis on  interpretation was placed on CT values rather  than quantities AB standards in quan...
 Analyses   using Lot 0704042:    Simulated samples & mixtures    Reproducibility    Reduced reaction volume    Sensi...
 Dr. Allison Eastman, Dr. Donald Orokos and  Dr. Sho Ya Wang NYS Police Forensic Investigation Center Kim and Melissa –...
Quantifiler-Y Validation Study
Quantifiler-Y Validation Study
Quantifiler-Y Validation Study
Quantifiler-Y Validation Study
Quantifiler-Y Validation Study
Upcoming SlideShare
Loading in …5
×

Quantifiler-Y Validation Study

1,150 views

Published on

My masters degree validation project with the New York State Police Forensic Investigation Center.

  • Be the first to comment

Quantifiler-Y Validation Study

  1. 1. Ashley Manning Hart University at Albany In partial fulfillment of theMaster of Science in Forensic Biology
  2. 2.  Extraction Quantitation Amplification STR DNA Analysis & Interpretation Report writing Review Testimony
  3. 3.  STR DNA analysis is very sensitive Inhibition of samples by either substrate or sample itself Wide range of amounts and quality of DNA at crime scene Best profiles from input DNA in concentration range 0.5 – 2ng/uL If too much DNA is added to amplification: stutter products, pull-up, -A peaks can be observed If too little DNA is added to amplification: allelic drop-out, allelic imbalance can be observed
  4. 4.  Crime scene samples can be “dirty” and contaminated with non-human DNA Microbial, plant, and/or animal DNA co-extract with human DNA recovered from the evidence FBI Quality Assurance Standard 9.3 requires laboratories to quantitate the amount of human DNA in crime scene evidence prior to subsequent DNA analysis Human STR DNA typing is optimized over a fairly narrow range of human DNA 4
  5. 5.  Ease of interpretation – especially with DNA mixtures Comparison purposes of evidence samples to reference samples Ability to upload to CODIS, NDIS Ability to be statistically meaningful Ability to stand up in court
  6. 6.  Known samples  Reference samples  Offender samples Questioned samples  Single source  Mixtures: male/male, male/female, female/female
  7. 7.  Quantifiler Y Human Male DNA Quantification Kit detects male DNA Most valuable when male:female DNA ratio low:  Body cavity swabs of female victim: oral, vaginal, anal  Bite marks, fingernail scrapings  When male cells cannot be separated from female cels Let’s explore the history of quantification first…
  8. 8.  UV Spectrometry Gel-based Methods Real-time PCR Methods
  9. 9.  Based on stacking of nitrogenous bases Stacking limits the amount of UV light absorbed Disadvantages:  Cannot distinguish dsDNA from ssDNA  Not human specific  RNA, protein and other contaminants also absorb these UV wavelengths  Not very sensitive  Relatively large amount of sample needed  Unacceptable method for crime scene evidence
  10. 10.  Gelsor blotting membranes utilized DNA specific dyes as reporter molecules  Species non-specific Advantages:  Broader quantitation range with lower limits than UV spectrometry  Possible to distinguish between dsDNA and ssDNA Disadvantages:  Interpretation can be subjective  Reporter molecules can be sensitive to chemicals in the solvent  Time-consuming – mostly hands-on
  11. 11.  Applied Biosystems method – chemiluminescence or chromogenic Slot blot on a nylon membrane HUMAN AND HIGHER PRIMATE Probe complimentary to alpha satellite DNA on chromosome 17 - D17S1 Disadvantages:  Very labor-intensive  Interpretation can be subjective  Low dynamic range – somewhat accurate over low concentration range
  12. 12.  Commonly used: Alu probes – more areas targeted as compared to slot blot methods Fluorescent probes Very sensitive, particularly for low copy number DNA Compatible with high-throughput DNA analysis systems Disadvantages:  “Home brew” kits must be quality assurance tested  Can be time-consuming
  13. 13.  Advantages  Less time-consuming than other methods  Less hands-on time  ability to be automated Disadvantages  Expensive kits  Expensive platform
  14. 14.  Targetshaploid 64-base length segment, Yp11.3 – the non-translated portion of the sex-determining region of the Y-chromosome (SRY) gene Contrast to Quantifiler Human Kit:  Targets diploid 62-base length segment, 5p15.33 – an intron of the human telomerase reverse transcriptase (hTERT) gene Bothkits utilize a TaqMan probe and a 5’ nuclease activity
  15. 15. -Two TaqMan probes target: untranslated region of SRY gene andIPC- Reporter dye (FAM for DNA of interest, VIC for IPC) on 5’ end ofprobe- MGB = minor groove binder – increases Tm  allows for shorterprobe- NRQ = non-flourescent quencher – proximity to reporter dyestops it from fluorescing until probe broken down-P = AmpliTaq Gold DNA polymerase
  16. 16. -PCR temperature increased to 72ºC  P moves 5’ – 3’ and extendsforward and reverse primers- Nucleotides (from Reaction Mix of kit) are added- Hybridized probe is broken up  reporter dye released and able tobe detected by camera in qPCR instrument
  17. 17.  PRIMER MIX: Human Target Specific Assay • FAM labeled probe (blue reporter dye) – targets Yp11.3 Internal Positive Control (IPC) • VIC labeled probe (green reporter dye) • 10,000 copies/rxn. of synthetic, non-human template • Inhibitor Detection TaqMan 2X Universal PCR Master Mix: PCR Reagents Passive Reference Dye • ROX (red reporter dye) – equalizes fluorescence
  18. 18.  Human male DNA, 200ng/ L – supplied with kit Standard curve: 50ng/ L to 23pg/ L Comparison of Q sample fluorescence to standards’ fluorescence Quality of standard curve is critical for estimations of quantities of human DNA
  19. 19.  96-well optical plate 7500 Real-Time PCR instrument with Sequence Detection Software 3 phases of PCR: geometric (exponential), linear & plateau qPCR measures fluorescence in real-time at geometric phase
  20. 20.  Fluorescenceemitted by release of reporter dye in sample  detected by camera Fluorescencethreshold set by Applied Biosystems’ software at 0.2 Amplificationcurve of sample crosses this threshold after a certain number of PCR cycles Thisintersection point is the Cycle Threshold (CT) for the sample
  21. 21.  Varies depending on amount of DNA in sample: well with more DNA will have a lower CT because it takes fewer cycles for that sample to reach the fluorescence threshold CT values are most accurate and reliable measurements of quantity of DNA in a sample
  22. 22.  Validation: to recognize, establish, or illustrate the worthiness or legitimacy of Validation of new techniques is essential and legally required in forensic labs Before Quantifiler Y Kit is implemented in a lab, tests must be completed to ensure its validity in the forensic community Validated by manufacturer, but manufacturer not held to same rigorous standards Definition from Merriam Webster Dictionary
  23. 23.  FBI Quality Assurance Standard 8.1.3  8.1.3.1 – Testing of known and non-probative evidence samples & Documentation of reproducibility and precision  8.1.3.3 – Qualifying tests administered to analysts prior to use SWGDAM Revised Validation Guidelines Standard 3  3.1 – Known and non-probative evidence samples  3.2 - Reproducibility and precision  3.4 - Sensitivity studies  3.5 - Mixture studies
  24. 24.  DNA extracts from simulated evidence Quantities and CT values compared Comparisons to the Amelogenin locus results obtained from STR DNA analysis Sample Number Sample Source 1 JRK T1 Blood card 2 JRK T1 Blood card 2 JRK T2 Buccal swab 3 JRK T2 Buccal swab JRK 1.1 Blood JRK 1.2 Buccal swab JRK 1.3 Blood JRK 2.1 Bloodstain JRK 2.2 Gum JRK 2.3 Buccal swab JRK 4.2 Buccal swab JRK 4.3 Bloodstain JRK 4.4 Buccal swab LMM 4.6 Buccal swab on cotton AMH 1 Buccal swab
  25. 25.  QuantiBlot performed by other analysts Quantifiler and Quantifiler Y combined on one assay
  26. 26. Sample Percentage Male DNA Electropherogram Quantifiler YJRK 5.5 NSF 5.57 7.16JRK 5.6 NSF 8.84 14.88JRK 4.8 NSF 1.34 0.716
  27. 27. CT Sample CT IPC CT Sample CT IPCSample Type Quantifiler Quantifiler Quantifiler Quantifiler Y Y Male 23.47 23.98 29.53 35.05 Standard 26.946 Female 23.02 N/A 28.83 33.74 Standard 27.018 Mixture 22.80 25.28 28.67 33.32 Standard 26.899
  28. 28.  Using Quantifiler Y Kit, many slopes fell below AB’s recommended range (-3.0 to -3.6) Erratic slope patterns using lot number 0704042
  29. 29. -Log of concentration plotted against CT values- Slope outside of acceptable, AB-validated range  overestimate ofDNA concentration to the left of the y-axis OR underestimate of DNAconcentration to the right of the y-axis- But, CT values are independent of concentration estimates made bythe software- Therefore, CT values were preferred for interpretation
  30. 30.  Issues not resolved so emphasis on interpretation was placed on CT values rather than quantities AB standards in quantification kits have been known to vary in concentration as much as ±50 – 100% with latest quality control tests allow for ±33% variation So… their QC standards may be more lax than would be preferred in the forensic community Kits with new lot numbers may yield different results
  31. 31.  Analyses using Lot 0704042:  Simulated samples & mixtures  Reproducibility  Reduced reaction volume  Sensitivity  Inhibition Many of the same samples analyzed again using Lot 0706043
  32. 32.  Dr. Allison Eastman, Dr. Donald Orokos and Dr. Sho Ya Wang NYS Police Forensic Investigation Center Kim and Melissa – the Queens of Quality Jeanette Kovari & Lisa McNab Amanda Brinton Jay Caponera Danielle Brownell

×