Uz. Dr. Ömer MÜSLÜMANOĞLU DNA Yapıtaşı Bazlar Bazlar Şekerler DNA-RNA arasında temel farklar DNA RNA Şeker deoksiriboz riboz Baz çifti Timin-Adenin Sitozin-Guanin Urasil-Adenin Sitozin-Guanin Yapısı Çift sarmal a heliks Tek Sarmal Düzensiz Dayanıklılık Stabil DNAse ile yıkılır Baz hidrolizine açık RNAse ile yıkılır Fonksyonu Genetik bilgiyi nukleusta saklar Genetik bilgiyi sitoplazmaya taşır SANTRAL DOĞMA replikasyon işlenme transkripsiyon translasyon DNA ISOLATION Sources of Biological Evidence • • • • • • • • Blood Semen Saliva Urine Hair Teeth Bone Tissue Blood stain Only a very small amount of blood is needed to obtain a DNA profile ORGANIC FTA Paper CHELEX SDS, DTT, EDTA and Blood stain proteinase K Blood stain Apply blood to paper and allow stain to dry Water INCUBATE (56 oC) PUNCH Centrifuge INCUBATE (ambient) Phenol, chloroform, isoamyl alcohol Centrifuge REMOVE supernatant VORTEX Centrifuge 5% Chelex TRANSFER aqueous (upper) phase to new tube REMOVE supernatant INCUBATE (56 oC) TE buffer CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation) Centrifuge QUANTITATE DNA WASH Multiple Times with extraction buffer PCR Reagents INCUBATE (100 oC) Centrifuge QUANTITATE DNA (NO DNA QUANTITATION TYPICALLY PERFORMED WITH UNIFORM SAMPLES) PERFORM PCR PERFORM PCR PERFORM PCR Figure 3.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Remove a portion of the mixed stain SDS, EDTA and proteinase K (cell lysis buffer) Incubate at 37 oC Centrifuge Perpetrator’s sperm mixed with victim’s epithelial cells sperm pellet SDS, EDTA and proteinase K + DTT “Male Fraction” REMOVE supernatant DTT lyses sperm heads sperm pellet “Female Fraction” Figure 3.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Differential extraction used to separate sperm (male fraction) from vaginal epithelial cells (female fraction) female male male female Evidence (female fraction) Evidence (male fraction) Suspect Victim The four samples typically associated with a forensic DNA case… QUANTITATION Unknown Samples Calibration standards 20 ng 10 ng 5 ng 2.5 ng 1.25 ng 0.63 ng Calibration standards ~2.5 ng 0.63 ng 1.25 ng 2.5 ng 5 ng 10 ng 20 ng Figure 3.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Calculation of the quantity of DNA in a cell 1. Molecular Weight of a DNA Basepair = 618g/mol A =: 313 g/mol; T: 304 g/mol; A-T base pairs = 617 g/mol G = 329 g/mol; C: 289 g/mol; G-C base pairs = 618 g/mol 2. Molecular weight of DNA = 1.85 x1012 g/mol There are 3 billion base pairs in a haploid cell ~3 x 109 bp (~3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol 3. Quantity of DNA in a haploid cell = 3 picograms 1 mole = 6.02 x 1023 molecules (1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules) = 3.08 x 10-12 g = 3.08 picograms (pg) A diploid human cell contains ~6 pg genomic DNA 4. One ng of DNA contains the DNA from 167 Importance of DNA Quantitation (prior to multiplex PCR) DNA amount (log scale) 100 ng High levels of DNA create interpretation challenges (more artifacts to review) -A Too much DNA Off-scale peaks Split peaks (+/-A) Locus-to-locus imbalance +A 10 ng 2.0 ng 1 ng Well-balanced STR multiplex STR Kits Work Best in This Range 0.5 ng 0.1 ng 0.01 ng Too little DNA Heterozygote peak imbalance Allele drop-out low Locus-to-locus imbalance Stochastic effect when amplifying levels of DNA produces allele dropout 6 bp deletion X Y X = 106 bp Y = 112 bp AmpFlSTR kits and PowerPlex 16 X = 212 bp Y = 218 bp PowerPlex 1.1 Female: X, X 1:1 Mixture: 3X + 1Y Male: X, Y Figure 5.11, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press PCR POLİMORFİZM • Bir popülasyonda mevcut olan genetik çeşitliliğe polimorfizm denir • DNA Polimorfizmi, DNA üzerinde hastalığa neden olmayan, suskun nükleotid değişimleri olarak tanımlanır. (A) Sequence polymorphism --------AGACTAGACATT--------------AGATTAGGCATT------- (B) Length polymorphism ---------(AATG)(AATG)(AATG)---------3 repeats ---------(AATG)(AATG)---------2 repeats Figure 2.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Chromosome 12 telomere p (short arm) Band 3 12p3 centromere q (long arm) Band 5 12q5 telomere Figure 2.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press İnsan Genomunda Dizi Tiplerinin Dağılımı Minisatellite Marker (D1S80) Flanking regions Repeat region GAGGACCACCAGGAAG 16 bp repeat unit STR Marker (TH01) Flanking regions Repeat region TCAT 4 bp repeat unit Figure 5.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Locus A Allele 1 Allele 2 4 Homologous pair of chromosomes 5 Allele 2 Allele 1 3 Homologous pair of chromosomes 6 Locus B Figure 2.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press 1 2 3 4 5 6 5’-TTTCCC TCAT TCAT TCAT TCAT TCAT TCAT TCACCATGGA-3’ 3’-AAAGGG AGTA AGTA AGTA AGTA AGTA AGTA AGTGGTACCT-5’ 6 5 4 3 2 1 Figure 5.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Temperature 94 oC 72 oC 72 oC 60 oC 94 oC 94 oC 94 oC 60 oC 72 oC 60 oC Single Cycle Time Typically 25-35 cycles performed during PCR The denaturation time in the first cycle is lengthened to ~10 minutes when using AmpliTaq Gold to perform a “hot-start” PCR Figure 4.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press PCR (A) Agarose yield gel results Smear of degraded DNA fragments High molecular weight DNA in a tight band (B) Degraded DNA sample D5S818 Good quality Degraded DNA DNA D13S317 D7S820 D16S539 CSF1PO Penta D Figure 7.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press (A) miniSTRs: new tool for degraded DNA Smaller PCR products work better with low Conventional PCR primer miniSTR primer copy number or fragmented DNA templates STR repeat region miniSTR primer Conventional PCR primer (B) Conventional STR test (COfiler™ kit) 150 bp smaller MiniSTR assay (using Butler et al. 2003 primers) Figure 7.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Y-STR Lineage Markers Autosomal Y-Chromosome Mitochondrial (passed on in part, from all ancestors) (passed on complete, but only by sons) (passed on complete, but only by daughters) Figure 9.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Female-Male Mixture Performance with Autosomal vs. Y-Chromosome DNA Markers No signal observed Female Victim DNA Profile Male Perpetrator DNA Profile DNA Profile from Crime Scene Autosomal STR Profile Y-Chromosome STR Profile Figure 9.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Modern Use of Y-STR Testing Captured December 13, 2003 Matching Y-STR Haplotype Used to Confirm Identity (along with allele sharing from autosomal STRs) Uday and Qusay Hussein Is this man really Sadaam Hussein? Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Box 23.1, p. 534 Killed July 22, 2003 PCR product size (bp) Figure A7.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press (A) Y-PLEX 6 (FAM-labeled loci) PCR product size (bp) Figure A7.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press ELEKTROFOREZ Elektroforez • Nükleik asitler (-) yüke sahitir (PO4) • Jelde göç etmeleri büyüklükleri ve yapıları ile ilgilidir % range % range agarose (kb) acrylamide (bp) 0.7 0.8-10 3.5 100-1000 0.9 0.5-7 5.0 80-500 1.2 0.4-6 8.0 60-400 1.5 0.2-4 12.0 40-200 2.0 0.1-3 20.0 10-100 Laser Capillary filled with polymer solution (cathode) Inlet Buffer 5-20 kV Detection window + (anode) Outlet Buffer Data Acquisition Sample tray Sample tray moves automatically beneath the cathode end of the capillary to deliver each sample in succession Figure 12.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press CAPİLLER ELECTROPHORESİS Cycseqpc.exe DNA PROFİLİ The Same 13 Locus STR Profile in Different Populations 1 in 837 trillion 1 in 0.84 quadrillion (1015) in U.S. Caucasian population (NIST) 1 in 2.46 quadrillion (1015) in U.S. Caucasian population (FBI)* 1 in 1.86 quadrillion (1015) in Canadian Caucasian population* 1 in 16.6 quadrillion (1015) in African American population (NIST) 1 in 17.6 quadrillion (1015) in African American population (FBI)* 1 in 18.0 quadrillion (1015) in U.S. Hispanic population (NIST) These values are for unrelated individuals assuming no population substructure (using only p2 and 2 pq) NIST study: Butler, J.M., et al. (2003) Allele frequencies for 15 autosomal STR loci on U.S. Caucasian, African American, and Hispanic populations. J. Forensic Sci. 48(4):908-911. (http://www.cstl.nist.gov/biotech/strbase/NISTpop.htm) *http://www.csfs.ca/pplus/profiler.htm Deciphering Artifacts from the True Alleles Biological (PCR) artifacts STR alleles Stutter products spike 6.0% 7.8% Dye blob Blue channel D3S1358 Incomplete adenylation +A +A -A -A D8S1179 stutter Green channel Pull-up (bleed-through) Yellow channel Red channel Figure 15.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press 100% (a) 85% Heterozygous peak region >70% MIXTURE REGION Stutter region <15% 9% 100% (b) >70% 60% Higher than typical stutter product (>15%) 25% Smaller peak area than normally seen with heterozygote partner alleles(<70%) 10% <15% Wrong side of allele to be typical stutter product Figure 7.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press DNA Size (bp) X Y RFUs C B A amelogenin 3 peaks at D8S1179 X-Y peak imbalance B A C D 4 peaks at D21S11 A B CD 4 peaks at D18S51 Figure 7.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press mtDNA Human Genome 23 Pairs of Chromosomes + mtDNA Located in cell nucleus http://www.ncbi.nlm.nih.gov/genome/guide/ Autosomes 2 copies per cell Located in mitochondria (multiple copies in cell cytoplasm) mtDNA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Nuclear DNA 3.2 billion bp Y Sexchromosomes 16,569 bp Mitochondrial DNA 100s of copies per cell Figure 2.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press 16024 16365 1 73 340 HV2 HV1 576 Control region (D-loop) 16024 OH F 12S rRNA T Heavy (H) strand cyt b 22 tRNAs 2 rRNAs V 13 genes 16S rRNA 1/16,569 P E ND6 L1 ND5 ND1 L2 S2 H “16,569” bp Light (L) strand ND4 OL Q I M A N ND2 C Y W ND4L R ND3 9-bp deletion S1 COI G COIII ATP6 ATP8 COII D K Figure 10.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press DNA DİZİ ANALİZİ Cycseqpc.exe 1 3 4 5 C D B 9 10 C 6 MtDNA Haplotype Groups: 1 2,3,6,8,11,13,15,16 4,9,10 5 7 12 14,17,18 7 B 11 C B 2 A E 12 B 15 B 8 B 13 B F 16 14 17 B G 18 G G Figure 10.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Markers Used (Biology) High Power of Discrimination (Genetics) RFLP Multi-Locus Probes Multiplex STRs RFLP Single Locus Probes mtDNA Low Slow PolyMarker D1S80 single STR DQa ABO blood groups Speed of Analysis (Technology) Fast Figure 1.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Lineage Markers Autosomal Y-Chromosome Mitochondrial (passed on in part, from all ancestors) (passed on complete, but only by sons) (passed on complete, but only by daughters) Figure 9.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Performed separately and preferably after evidence is completed Extract mtDNA from evidence (Q) sample Extract mtDNA from reference (K) sample PCR Amplify HV1 and HV2 Regions PCR Amplify HV1 and HV2 Regions Sequence HV1 and HV2 Amplicons Sequence HV1 and HV2 Amplicons (both strands) (both strands) Confirm sequence with forward and reverse strands Confirm sequence with forward and reverse strands Note differences from Anderson (reference) sequence Note differences from Anderson (reference) sequence Compare Q and K sequences Compare with database to determine haplotype frequency Figure 10.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Roche (F15975) FBI A1 (L15997) GAAAAAGTCT TTAACTCCAC CATTAGCACC CAAAGCTAAG ATTCTAATTT AAACTATTCT CTTTTTCAGA AATTGAGGTG GTAATCGTGG GTTTCGATTC TAAGATTAAA TTTGATAAGA 15970 15980 15990 16000 16010 16020 HV1 CTGTTCTTTC ATGGGGAAGC AGATTTGGGT ACCACCCAAG TATTGACTCA CCCATCAACA GACAAGAAAG TACCCCTTCG TCTAAACCCA TGGTGGGTTC ATAACTGAGT GGGTAGTTGT 16030 16040 16050 16060 16070 Hypervariable Region I 16024-16365 342 bp examined 16080 Roche IA 16093 C 16126 C A 16129 ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT TGGCGATACA TAAAGCATGT AATGACGGTC GGTGGTACTT ATAACATGCC ATGGTATTTA 16090 16100 16110 16120 16130 16140 HVI C-stretch ACTTGACCAC CTGTAGTACA TAAAAACCCA ATCCACATCA AAACCCCCTC CCCATGCTTA TGAACTGGTG GACATCATGT ATTTTTGGGT TAGGTGTAGT TTTGGGGGAG GGGTACGAAT 16150 16160 16170 16180 16190 16200 CAAGCAAGTA CAGCAATCAA CCCTCAACTA TCACACATCA ACTGCAACTC CAAAGCCACC GTTCGTTCAT GTCGTTAGTT GGGAGTTGAT AGTGTGTAGT TGACGTTGAG GTTTCGGTGG 16210 16220 16230 16240 16250 16260 Roche IC Roche IE T T C G C CCTCACCCAC TAGGATACCA ACAAACCTAC CCACCCTTAA CAGTACATAG TACATAAAGC GGAGTGGGTG ATCCTATGGT TGTTTGGATG GGTGGGAATT GTCATGTATC ATGTATTTCG 16270 16280 16290 16300 16310 16320 Roche ID C HV1 CATTTACCGT ACATAGCACA TTACAGTCAA ATCCCTTCTC GTCCCCATGG ATGACCCCCC GTAAATGGCA TGTATCGTGT AATGTCAGTT TAGGGAAGAG CAGGGGTACC TACTGGGGGG 16330 16340 16350 16360 16370 16380 TCAGATAGGG GTCCCTTGAC CACCATCCTC CGTGAAATCA ATATCCCGCA CAAGAGTGCT AGTCTATCCC CAGGGAACTG GTGGTAGGAG GCACTTTAGT TATAGGGCGT GTTCTCACGA 16390 16400 FBI B1 (H16391) 16410 16420 Roche (R16418) 16430 16440 SSO Probes 16093 16126 16129 16270 16278 16304 16309 16311 16362 Only 9 sites examined (A) mtDNA Sequences Aligned with rCRS (positions 16071-16140) 16090 16100 16110 16120 16130 16140 rCRS ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT Q ACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT K ACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT (B) Reporting Format with Differences from rCRS Sample Q 16093C 16129A Sample K 16093C 16129A Figure 10.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press • İnsan genomu 3,164,700,000 nukleotidden oluşmaktadır. • Toplam gen sayısı 29,000-36,000 arasındadır. • Nükleotid dizilerinin %99’u bütün insanlarda aynıdır. • Bu güne kadar insanda 1,5 milyon kadar tek nukleotid değişikliği bölgesi saptanmıştır. • Tanımlanmış genlerin %50’den fazlasının işlevleri henüz bilinmemektedir. • Genomun yaklaşık %2’si proteinleri kodlamaktadır. • Proteinleri kodlamayan dizi tekrarları, genomun büyük bölümünü oluşturur. İnsan Genomundan Beklentilerimiz • Moleküler Tıp -Tanı yöntemlerinin geliştirilmesi - Hastalıklara genetik yatkınlığın belirlenmesi - Genetik yapıya özgü ilaçlar geliştirilmesi - Gen tedavisi yöntemlerinin geliştirilmesi • Biyoarkeoloji, Antropoloji ve Tarih - Değişik toplumların göç yollarının ve akrabalıklarının araştırılması - Y kromozom mutasyonlarının incelenmesiyle erkek dağılımının ve göçlerin araştırılması • DNA Tanımlama - Adli tıpta suçluların belirlenmesi - Kan bağlarının saptanması - Organ nakillerinde doku uyumunun kesin şekilde saptanması - Soy ağaçlarının geliştirilmesi Kuşkular • Genetik Bilginin Özelliği ve Gizliliği - Genetik bilgiye kim sahip olacak ve kontrol edecek? - Genetik bilgilerin gizliliği tıbbi gizlilikten farklı mı? • Genetik bilginin kullanılması - Bireye ait genetik bilgilere kim ulaşabilecek ve bu bilgileri nasıl kullanacak? SABRINIZ İÇİN TEŞEKKÜRLER