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Uz. Dr. Ömer MÜSLÜMANOĞLU. DNA Yapıtaşı Bazlar.

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... konulu sunumlar: "Uz. Dr. Ömer MÜSLÜMANOĞLU. DNA Yapıtaşı Bazlar."— Sunum transkripti:

1 Uz. Dr. Ömer MÜSLÜMANOĞLU

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3 DNA Yapıtaşı

4 Bazlar

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6 Şekerler

7 DNA-RNA arasında temel farklar DNARNA Şekerdeoksiribozriboz Baz çiftiTimin-Adenin Sitozin-Guanin Urasil-Adenin Sitozin-Guanin YapısıÇift sarmal  heliks Tek Sarmal Düzensiz DayanıklılıkStabil DNAse ile yıkılır Baz hidrolizine açık RNAse ile yıkılır FonksyonuGenetik bilgiyi nukleusta saklar Genetik bilgiyi sitoplazmaya taşır

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9 replikasyon transkripsiyon işlenme translasyon SANTRAL DOĞMA

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13 DNA ISOLATION

14 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

15 ORGANIC FTA Paper CHELEX Blood stain PUNCH WASH Multiple Times with extraction buffer PERFORM PCR PCR Reagents SDS, DTT, EDTA and proteinase K INCUBATE (56 o C) Phenol, chloroform, isoamyl alcohol QUANTITATE DNA Apply blood to paper and allow stain to dry Blood stain VORTEX (NO DNA QUANTITATION TYPICALLY PERFORMED WITH UNIFORM SAMPLES) Water INCUBATE (ambient) 5% Chelex INCUBATE (100 o C) REMOVE supernatant INCUBATE (56 o C) QUANTITATE DNA PERFORM PCR Centrifuge REMOVE supernatant TRANSFER aqueous (upper) phase to new tube CONCENTRATE sample (Centricon/Microcon-100 or ethanol precipitation) Centrifuge TE buffer Figure 3.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

16 Perpetrator’s sperm mixed with victim’s epithelial cells Centrifuge REMOVE supernatant SDS, EDTA and proteinase K (cell lysis buffer) Remove a portion of the mixed stain SDS, EDTA and proteinase K + DTT Incubate at 37 o C sperm pellet DTT lyses sperm heads “Male Fraction” “Female Fraction” sperm pellet Figure 3.2, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

17 Evidence (female fraction) Evidence (male fraction) Suspect Victim Differential extraction used to separate sperm (male fraction) from vaginal epithelial cells (female fraction) male female male female The four samples typically associated with a forensic DNA case…

18 QUANTITATION

19 20 ng 10 ng 5 ng 2.5 ng 1.25 ng 0.63 ng20 ng 10 ng 5 ng 2.5 ng 1.25 ng 0.63 ng Calibration standards Unknown Samples ~2.5 ng Figure 3.3, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

20 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 x10 12 g/mol There are 3 billion base pairs in a haploid cell ~3 x 109 bp (~3 x 10 9 bp) x (618 g/mol/bp) = 1.85 x 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 g = 3.08 picograms (pg) A diploid human cell contains ~6 pg genomic DNA 4. One ng of DNA contains the DNA from 167 diploid cells 1 ng genomic DNA (1000 pg)/6pg/cell = ~333 copies of each locus (2 per 167 diploid genomes)

21 Importance of DNA Quantitation (prior to multiplex PCR) DNA amount (log scale) 0.5 ng -A +A Too much DNA  Off-scale peaks  Split peaks (+/-A)  Locus-to-locus imbalance 100 ng 10 ng 1 ng 0.1 ng 0.01 ng 2.0 ng Too little DNA  Heterozygote peak imbalance  Allele drop-out  Locus-to-locus imbalance Stochastic effect when amplifying low levels of DNA produces allele dropout STR Kits Work Best in This Range High levels of DNA create interpretation challenges (more artifacts to review) Well-balanced STR multiplex

22 X Y 6 bp deletion Female: X, X Male: X, Y 1:1 Mixture: 3X + 1Y X = 212 bp Y = 218 bp X = 106 bp Y = 112 bp AmpFlSTR kits and PowerPlex 16 PowerPlex 1.1 Figure 5.11, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

23 PCR

24 POLİMORFİZM Bir popülasyonda mevcut olan genetik çeşitliliğe polimorfizm denir DNA Polimorfizmi, DNA üzerinde hastalığa neden olmayan, suskun nükleotid değişimleri olarak tanımlanır.

25 2 repeats 3 repeats AGACTAGACATT AGATTAGGCATT (AATG)(AATG)(AATG) (AATG)(AATG) (B) Length polymorphism (A) Sequence polymorphism Figure 2.5, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

26 p (short arm) centromere telomere q (long arm) telomere Band 5 Band 3 Chromosome 12 12p3 12q5 Figure 2.4, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

27 İnsan Genomunda Dizi Tiplerinin Dağılımı

28 Minisatellite Marker (D1S80) GAGGACCACCAGGAAG Repeat region Flanking regions 16 bp repeat unit STR Marker (TH01) TCAT Repeat region Flanking regions 4 bp repeat unit Figure 5.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

29 Homologous pair of chromosomes Locus A Locus B Allele 1 Allele 2 Allele 1 Figure 2.6, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

30 ’-TTTCCC TCAT TCAT TCAT TCAT TCAT TCAT TCACCATGGA-3’ 3’-AAAGGG AGTA AGTA AGTA AGTA AGTA AGTA AGTGGTACCT-5’ Figure 5.2, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

31 94 o C 60 o C 72 o C Time Temperature Single Cycle Typically cycles performed during PCR 94 o C 60 o C 72 o C 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, 2 nd Edition © 2005 Elsevier Academic Press

32 PCR

33 Degraded DNA sample D5S818 D13S317 D7S820 D16S539 CSF1PO Penta D Agarose yield gel results Smear of degraded DNA fragments High molecular weight DNA in a tight band (A) (B) Good quality DNA Degraded DNA Figure 7.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

34 STR repeat region miniSTR primer Conventional PCR primer (A) (B) Conventional STR test (COfiler™ kit) MiniSTR assay (using Butler et al primers) Figure 7.2, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press Smaller PCR products work better with low copy number or fragmented DNA templates miniSTRs: new tool for degraded DNA 150 bp smaller

35 Y-STR

36 Autosomal (passed on in part, from all ancestors) Y-Chromosome (passed on complete, but only by sons) Mitochondrial (passed on complete, but only by daughters) Lineage Markers Figure 9.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

37 Female-Male Mixture Performance with Autosomal vs. Y-Chromosome DNA Markers Female Victim DNA Profile Male Perpetrator DNA Profile DNA Profile from Crime Scene Autosomal STR Profile Y-Chromosome STR Profile No signal observed Figure 9.2, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

38 Modern Use of Y-STR Testing Captured December 13, 2003 Is this man really Sadaam Hussein? Uday and Qusay Hussein Killed July 22, 2003 Matching Y-STR Haplotype Used to Confirm Identity (along with allele sharing from autosomal STRs) Butler, J.M. (2005) Forensic DNA Typing, 2 nd Edition, Box 23.1, p. 534

39 PCR product size (bp) Figure A7.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

40 PCR product size (bp) (A) Y-PLEX 6 (FAM-labeled loci) Figure A7.2, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

41 ELEKTROFOREZ

42 Nükleik asitler (-) yüke sahitir (PO 4 ) Jelde göç etmeleri büyüklükleri ve yapıları ile ilgilidir Elektroforez

43 Laser Inlet Buffer Capillary filled with polymer solution 5-20 kV -+ Outlet Buffer Sample tray Detection window (cathode) (anode) Data Acquisition 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, 2 nd Edition © 2005 Elsevier Academic Press

44 CAPİLLER ELECTROPHORESİS

45 DNA PROFİLİ

46 The Same 13 Locus STR Profile in Different Populations 1 in 0.84 quadrillion (10 15 ) in U.S. Caucasian population (NIST) 1 in 2.46 quadrillion (10 15 ) in U.S. Caucasian population (FBI)* 1 in 1.86 quadrillion (10 15 ) in Canadian Caucasian population* 1 in 16.6 quadrillion (10 15 ) in African American population (NIST) 1 in 17.6 quadrillion (10 15 ) in African American population (FBI)* 1 in 18.0 quadrillion (10 15 ) in U.S. Hispanic population (NIST) *http://www.csfs.ca/pplus/profiler.htm 1 in 837 trillion These values are for unrelated individuals assuming no population substructure (using only p 2 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): (http://www.cstl.nist.gov/biotech/strbase/NISTpop.htm)

47 Dye blob STR alleles stutter Pull-up (bleed-through) spike Blue channel Green channel Yellow channel Red channel Figure 15.4, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press D3S1358 Stutter products 6.0%7.8% Incomplete adenylation D8S1179 -A +A -A +A Biological (PCR) artifacts Deciphering Artifacts from the True Alleles

48 <15% Stutter region >70% 100% Heterozygous peak region 85% MIXTURE REGION 9% Higher than typical stutter product (>15%) 100% <15% >70% 60% 10% 25% Wrong side of allele to be typical stutter product Smaller peak area than normally seen with heterozygote partner alleles(<70%) (a) (b) Figure 7.3, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

49 amelogenin X-Y peak imbalance A B C B A C D D C B A 3 peaks at D8S peaks at D21S11 4 peaks at D18S51 X Y DNA Size (bp) RFUs Figure 7.6, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

50 mtDNA

51 X Y Human Genome 23 Pairs of Chromosomes + mtDNA Sex- chromosomes mtDNA 16,569 bp Autosomes Mitochondrial DNA Nuclear DNA 3.2 billion bp Located in cell nucleus Located in mitochondria (multiple copies in cell cytoplasm) 2 copies per cell 100s of copies per cell Figure 2.3, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

52 Heavy (H) strand Light (L) strand Figure 10.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

53 DNA DİZİ ANALİZİ

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55 MtDNA Haplotype Groups: 1 2,3,6,8,11,13,15,16 4,9, ,17,18 MtDNA Haplotype Groups: 1 2,3,6,8,11,13,15,16 4,9, ,17,18 A B B C C C D B B B B B B E F G G G Figure 10.2, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

56 Speed of Analysis (Technology) Power of Discrimination (Genetics) Low High SlowFast Markers Used (Biology) RFLP Single Locus Probes RFLP Multi-Locus Probes ABO blood groups Multiplex STRs DQ  single STR D1S80 mtDNA PolyMarker Figure 1.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

57 Autosomal (passed on in part, from all ancestors) Y-Chromosome (passed on complete, but only by sons) Mitochondrial (passed on complete, but only by daughters) Lineage Markers Figure 9.1, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

58 Compare with database to determine haplotype frequency Extract mtDNA from evidence (Q) sample PCR Amplify HV1 and HV2 Regions Sequence HV1 and HV2 Amplicons (both strands) Confirm sequence with forward and reverse strands Note differences from Anderson (reference) sequence Compare Q and K sequences Performed separately and preferably after evidence is completed Extract mtDNA from reference (K) sample PCR Amplify HV1 and HV2 Regions Sequence HV1 and HV2 Amplicons (both strands) Confirm sequence with forward and reverse strands Note differences from Anderson (reference) sequence Figure 10.4, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

59 GAAAAAGTCT TTAACTCCAC CATTAGCACC CAAAGCTAAG ATTCTAATTT AAACTATTCT CTTTTTCAGA AATTGAGGTG GTAATCGTGG GTTTCGATTC TAAGATTAAA TTTGATAAGA CTGTTCTTTC ATGGGGAAGC AGATTTGGGT ACCACCCAAG TATTGACTCA CCCATCAACA GACAAGAAAG TACCCCTTCG TCTAAACCCA TGGTGGGTTC ATAACTGAGT GGGTAGTTGT C C A ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT TGGCGATACA TAAAGCATGT AATGACGGTC GGTGGTACTT ATAACATGCC ATGGTATTTA ACTTGACCAC CTGTAGTACA TAAAAACCCA ATCCACATCA AAACCCCCTC CCCATGCTTA TGAACTGGTG GACATCATGT ATTTTTGGGT TAGGTGTAGT TTTGGGGGAG GGGTACGAAT CAAGCAAGTA CAGCAATCAA CCCTCAACTA TCACACATCA ACTGCAACTC CAAAGCCACC GTTCGTTCAT GTCGTTAGTT GGGAGTTGAT AGTGTGTAGT TGACGTTGAG GTTTCGGTGG T T C G C CCTCACCCAC TAGGATACCA ACAAACCTAC CCACCCTTAA CAGTACATAG TACATAAAGC GGAGTGGGTG ATCCTATGGT TGTTTGGATG GGTGGGAATT GTCATGTATC ATGTATTTCG C CATTTACCGT ACATAGCACA TTACAGTCAA ATCCCTTCTC GTCCCCATGG ATGACCCCCC GTAAATGGCA TGTATCGTGT AATGTCAGTT TAGGGAAGAG CAGGGGTACC TACTGGGGGG TCAGATAGGG GTCCCTTGAC CACCATCCTC CGTGAAATCA ATATCCCGCA CAAGAGTGCT AGTCTATCCC CAGGGAACTG GTGGTAGGAG GCACTTTAGT TATAGGGCGT GTTCTCACGA FBI A1 (L15997) Roche (F15975) HV HVI C-stretch Roche IA Roche ID Roche IC Roche IE HV1 FBI B1 (H16391) Roche (R16418) Hypervariable Region I bp examined SSO Probes Only 9 sites examined

60 Sample Q 16093C 16129A Sample K 16093C 16129A ACCGCTATGT ATTTCGTACA TTACTGCCAG CCACCATGAA TATTGTACGG TACCATAAAT rCRS ACCGCTATGT ATCTCGTACA TTACTGCCAG CCACCATGAA TATTGTACAG TACCATAAAT Q K (A) mtDNA Sequences Aligned with rCRS (positions ) (B) Reporting Format with Differences from rCRS Figure 10.8, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition © 2005 Elsevier Academic Press

61 İnsan genomu 3,164,700,000 nukleotidden oluşmaktadır. Toplam gen sayısı 29,000-36,000 arasındadır. Nükleotid dizilerinin %99’u bütün insanlarda aynıdır.

62 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.

63 Moleküler TıpMoleküler Tıp -Tanı yöntemlerinin geliştirilmesi - Hastalıklara genetik yatkınlığın belirlenmesi - Genetik yapıya özgü ilaçlar geliştirilmesi -Gen tedavisi yöntemlerinin geliştirilmesi İnsan Genomundan Beklentilerimiz

64 Biyoarkeoloji, Antropoloji ve TarihBiyoarkeoloji, Antropoloji ve Tarih - Değişik toplumların göç yollarının ve akrabalıklarının araştırılması - Y kromozom mutasyonlarının incelenmesiyle erkek dağılımının ve göçlerin araştırılması

65 DNA TanımlamaDNA Tanımlama - Adli tıpta suçluların belirlenmesi - Kan bağlarının saptanması - Organ nakillerinde doku uyumunun kesin şekilde saptanması - Soy ağaçlarının geliştirilmesi

66 Kuşkular Genetik Bilginin Özelliği ve GizliliğiGenetik Bilginin Özelliği ve Gizliliği - Genetik bilgiye kim sahip olacak ve kontrol edecek? - Genetik bilgilerin gizliliği tıbbi gizlilikten farklı mı? Genetik bilginin kullanılmasıGenetik bilginin kullanılması - Bireye ait genetik bilgilere kim ulaşabilecek ve bu bilgileri nasıl kullanacak?

67 SABRINIZ İÇİN TEŞEKKÜRLER


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