Characters of degraded DNA Forensic samples are exposed to unstable - PDF document

STR Typing of Degraded DNA using a Repair Enzyme and Whole Genome Amplification Jeong Eun Sim 1 , Hwan Young Lee 1 , Seung Hwan Lee 2 , Woo Ick Yang 1 , Kyoung ‐ Jin Shin 1 1 Department of Forensic Medicine and Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea 2 DNA Analysis Lab., Div. of Forensic Science, Supreme Prosecutors' Office, Seoul, Korea Characters of degraded DNA • Forensic samples are exposed to unstable environments • DNA could be damaged  It may be degraded into fragments smaller than amplicon size  It may contain only small amounts of genomic DNA  Therefore, it may result in failure of PCR amplification at some of STR loci and produce incomplete DNA profile ⇒ MiniSTR: PCR amplicon size can be reduced Repair enzyme, Whole genome amplification The Korean Society for Legal Medicine

Repairing Enzyme • Within living cells, the integrity of DNA molecules is continually maintained by enzymatic repair processes. • By using repairing enzyme, DNA repair of living cell can be carried out in vitro. • Commercial reagent : PreCR Repair Mix  (Uracil ‐ DNA Glycosylase, formamidopyrimidine ‐ DNA glycosylase, Endonuclease IV, Endonuclease VIII, T4 Endonuclease V, Bst DNA Polymerase, Taq DNA ligase) • In previous studies, only UV damaged DNA and DNA from skeletal remains were evaluated. Repair enzyme should be evaluated in various types of damaged DNA and forensic samples The Korean Society for Legal Medicine Whole Genome Amplification (WGA) • WGA is a technique to specifically increase the DNA quantities originating from samples with limited DNA contents.  Preimplantation genetic diagnosis (PGD) • Isothermal WGA method  MDA (Multiple Displacement Amplification)  Random hexamer primer Hyperbranched Displacement Polymerization  structure Phi29 DNA polymerase Template DNA (http://www.qiagen.com) After repairing of degraded DNA, MDA based WGA method can be applied to repaired DNA. The Korean Society for Legal Medicine

Aims of This Study • Validation of the availability and efficacy of repairing enzyme in various types of degraded DNA • To present the possibility of application of WGA to forensic field • To increase the success rate of DNA typing in damaged sample The Korean Society for Legal Medicine Materials and Methods: Sample preparation • Artificially degraded DNA  9948 standard DNA, K562 high molecular DNA  UV radiated DNA: UV radiation during 2 minutes by CL ‐ 1000 Ultraviolet cross ‐ linker  Oxidized DNA: Hydroxyl radical by fenton reaction using Fe 2+ , H 2 O 2  Acid/heat damaged DNA: NaCl, Sodium ‐ Na, 70 ° C, 10 hours incubation  DNase I treated DNA: 0.006 unit DNase I treatment for 10~15 min • Naturally degraded DNA  Dried blood spot, dried saliva spot from 5 applicants: IRB approval, 10uL, sunshine exposure and air dry during 2 weeks – QIAamp DNA Investigator Kit  Old skeletal remains: over 60 years – DNA extracted by Lee et al . ( Forensic Sci Int Genet. 2010; In press) • All of the samples were prepared in quintuplicate. The Korean Society for Legal Medicine

Materials and Methods: Repair of degraded DNA Amplification of autosomal STR: AmpF ℓ STR Identifiler Kit • Reagents Repair Control 6.0 ㎕ 1.5 ㎕ 9.0 ㎕ 2.8 ㎕ dH 2 0 9.5 ㎕ 3.8 ㎕ 9.5 ㎕ 3.8 ㎕ Identifiler Reaction Mix 10 × NAD + 2.5 ㎕ 1.0 ㎕ ‐ ‐ AmpliTaq Gold DNA polymerase (5 unit/ ㎕ ) 0.5 ㎕ 0.4 ㎕ 0.5 ㎕ 0.4 ㎕ 0.5 ㎕ 0.3 ㎕ PreCR Repair Mix ‐ ‐ 1.0 ㎕ 1.0 ㎕ 1.0 ㎕ 1.0 ㎕ Demaged DNA Incubation at 37 ° C, 20 min for repairing ‐ ‐ 5.0 ㎕ 2.0 ㎕ 5.0 ㎕ 2.0 ㎕ Identifiler Primer Set 25.0 ㎕ 10.0 ㎕ 25.0 ㎕ 10.0 ㎕ Total reaction volume Thermal cycling for PCR amplification 95 ° C for 11 min; 94 ° C for 1 min: 59 ° C for 1 min: and 72 ° C for 1 min × 28 cycles; a final extension at 60 ° C for 60 min. In case of DNA obtained from skeletal remains, 3.5 U of Gold Taq enzyme and 2.0 uL of template DNA were used with 5 more amplification cycle. The Korean Society for Legal Medicine Materials and Methods: WGA • Sensitivity test: Serial dilution samples (10 ng, 1 ng, 500 pg, 250 pg, 125 pg, 62.5 pg, 31.3 pg) • Performing of WGA: GenomiPhi V2 Amplification Kit (MDA based WGA) • Quantification: TBS ‐ 380 Mini ‐ Fluorometer • Amplification of autosomal STR: AmpF ℓ STR Identifiler Kit (http://www.gelifesciences.com) The Korean Society for Legal Medicine

Materials and Methods: Combination of Repair and WGA • Whole volume of repaired DNA should be used for WGA • Combination of the repairing step and WGA Process Reagents Volume 5.7 ㎕ dH 2 0 10 × ThermoPol Reaction Buffer 1.0 ㎕ 1.0 ㎕ 1mM dNTP 10 × NAD + 1.0 ㎕ DNA repairing PreCR Repair Mix 0.3 ㎕ Demaged DNA (1 ng/ ㎕ ) 1.0 ㎕ 10.0 ㎕ Total reaction volume Incubation at 37 ° C, 20 min for repairing Reducing volume Vacuum dry during 15 min 95 ° C, 3 min denaturation, ice Add Reaction Buffer 9 ㎕ , Enzyme 1 ㎕ WGA Elongation: 30 ° C, 1.5 hour incubation Enzyme inactivation: 65 ° C, 10 min • Using naturally degraded DNA, applicability of combining step was evaluated . • Quantification: TBS ‐ 380 Mini ‐ Fluorometer • Amplification of autosomal STR: AmpF ℓ STR Identifiler Kit The Korean Society for Legal Medicine Results : Repair of Degraded DNA 3000 3000 2500 2500 Mean peak height Mean peak height × 4.0 2000 2000 UV radiated DNA 1500 1500 1000 1000 500 500 : Pyrimidine dimer 0 0 D8S1179 D8S1179 D21S11 D21S11 D7S820 D7S820 CSF1PO D3S1358 CSF1PO D3S1358 TH01 TH01 D13S317 D16S539 D2S1338 D19S433 D13S317 D16S539 D2S1338 D19S433 vWA vWA TPOX TPOX D18S51 D18S51 D5S818 D5S818 FGA FGA Loci Loci 7500 7500 1200 1200 6250 6250 Mean peak height Mean peak height 1000 1000 × 1.8 Oxidized DNA Mean peak height Mean peak height 5000 5000 800 800 3750 3750 600 600 2500 2500 400 400 : Guanine  8 ‐ oxo ‐ Guanine, 1250 1250 200 200 0 0 0 0 nick, fragmentation D8S1179 D8S1179 D21S11 D21S11 D7S820 D7S820 CSF1PO D3S1358 CSF1PO D3S1358 TH01 TH01 D13S317 D16S539 D2S1338 D19S433 D13S317 D16S539 D2S1338 D19S433 vWA vWA TPOX TPOX D18S51 D18S51 D5S818 D5S818 FGA FGA D8S1179 D8S1179 D21S11 D21S11 D7S820 D7S820 CSF1PO CSF1PO D3S1358 D3S1358 TH01 TH01 D13S317 D13S317 D16S539 D2S1338 D19S433 D16S539 D2S1338 D19S433 vWA vWA TPOX TPOX D18S51 D18S51 D5S818 D5S818 FGA FGA Loci Loci Loci Loci 6000 6000 7500 7500 5000 5000 Mean peak height Mean peak height 6250 6250 Mean peak height Mean peak height × 2.2 4000 4000 Acid/heat DNA 5000 5000 3000 3000 3750 3750 2000 2000 2500 2500 1000 1000 : Apurine/apyrimidine site, 1250 1250 0 0 0 0 D8S1179 D8S1179 D21S11 D21S11 D7S820 D7S820 CSF1PO CSF1PO D3S1358 D3S1358 TH01 TH01 D13S317 D13S317 D16S539 D16S539 D2S1338 D2S1338 D19S433 D19S433 vWA vWA TPOX TPOX D18S51 D18S51 D5S818 D5S818 FGA FGA deamination, D8S1179 D8S1179 D21S11 D21S11 D7S820 D7S820 CSF1PO D3S1358 CSF1PO D3S1358 TH01 TH01 D13S317 D16S539 D2S1338 D19S433 D13S317 D16S539 D2S1338 D19S433 vWA vWA TPOX TPOX D18S51 D18S51 D5S818 D5S818 FGA FGA Loci Loci Loci Loci nick, fragmentation Control Control Repair Repair 1200 1200 1000 1000 Mean peak height Mean peak height 800 800 DNase I treated DNA 600 600 400 400 200 200 : Nick, fragmentation 0 0 D8S1179 D8S1179 D21S11 D21S11 D7S820 D7S820 CSF1PO CSF1PO D3S1358 D3S1358 TH01 TH01 D13S317 D13S317 D16S539 D2S1338 D19S433 D16S539 D2S1338 D19S433 vWA vWA TPOX TPOX D18S51 D18S51 D5S818 D5S818 FGA FGA Loci Loci The Korean Society for Legal Medicine

Results : Repair of Degraded DNA 4000 4000 Mean peak height Mean peak height 3000 3000 × 1.3 Dried blood spot 2000 2000 1000 1000 ; UV damage, desiccation, 0 0 D8S1179 D8S1179 D21S11 D21S11 D7S820 D7S820 CSF1PO D3S1358 CSF1PO D3S1358 TH01 TH01 D13S317 D16S539 D2S1338 D19S433 D13S317 D16S539 D2S1338 D19S433 vWA vWA TPOX TPOX D18S51 D18S51 D5S818 D5S818 FGA FGA oxidation Loci Loci 2000 2000 Mean peak height Mean peak height 1500 1500 × 2.3 Dried saliva spot 1000 1000 500 500 0 0 ; UV damage, desiccation, D8S1179 D8S1179 D21S11 D21S11 D7S820 D7S820 CSF1PO D3S1358 CSF1PO D3S1358 TH01 TH01 D13S317 D16S539 D2S1338 D19S433 D13S317 D16S539 D2S1338 D19S433 vWA vWA TPOX TPOX D18S51 D18S51 D5S818 D5S818 FGA FGA oxidation Loci Loci 1600 1600 Mean peak height Mean peak height 1200 1200 800 800 Skeletal remains 400 400 0 0 Control Peak imbalance was D8S1179 D8S1179 D21S11 D21S11 D7S820 D7S820 CSF1PO D3S1358 CSF1PO D3S1358 TH01 TH01 D13S317 D16S539 D2S1338 D19S433 D13S317 D16S539 D2S1338 D19S433 vWA vWA TPOX TPOX D18S51 D18S51 D5S818 D5S818 FGA FGA Loci Loci decreased (50%) Control Control Repaired Repaired Non ‐ specific peak Repaired The Korean Society for Legal Medicine Results : Sensitivity Test of WGA • Mean concentration of amplified DNA by WGA in sensitivity test Input DNA for WGA 10 ng 1 ng 500 pg 250 pg 125 pg 62.5 pg 31.3 pg Concentrations (ng/uL) 191.9 179.8 148.5 112.7 34.7 46.4 18.8 Peak imbalance was shown 10 ng 10 ng for WGA for WGA • between loci 1 ng 1 ng • between alleles for WGA for WGA 500 pg 500 pg for WGA for WGA 250 pg 250 pg Allele drop out for WGA for WGA 125 pg 125 pg for WGA for WGA 62.5 pg 62.5 pg for WGA for WGA 31.3 pg 31.3 pg for WGA for WGA The Korean Society for Legal Medicine