Post on 18-Jan-2016
证据技术实验教学中心法医室
DNA 在法庭科学中的应用
DNA in the Cell
PCR 目的片段( Target Region )
PCR 目的片段( Target Region )
染色体chromosome
细胞核
cell nucleus
核苷
nucleotides
Projeto Genoma Humano
Sample Sequences
actgatAAAAAAAAGGGGGGGggcgtacacattagCTGATTCCAATACAGacgt
aaAAAAAAAGAGGGGGGaaacttttccgaataCTGATTCCAATACAGgatcagt
atgacttAAAAAAAAGGGGGGGtgctctcccgattttCCTGATTCCAATACAGc
aggAAAAAAAGAGGGGGGagccctaacggacttaatCCTGATTCCAATACAGta
ggaggAAAAAAAGAGGGGGGagccctaacggacttaatCCTGATTCCAATACAG
GGGACTCCGCGTCTCGCTCTCTGTGTTCCAATCGCCCGGTGCGGTGGTGCAGGGTCTCGGGCTAGTCATGGCGTCCCCGTCTCGGAGACTGCAGACTAAACCAGTCATTACTTGTTTCAAGAGCGTTCTGCTAATCTACACTTTTATTTTCTGGATCACTGGCGTTATCCTTCTTGCAGTTGGCATTTGGGGCAAGGTGAGCCTGGAGAATTACTTTTCTCTTTTAAATGAGAAGGCCACCAATGTCCCCTTCGTGCTCATTGCTACTGGTACCGTCATTATTCTTTTGGGCACCTTTGGTTGTTTTGCTACCTGCCGAGCTTCTGCATGGATGCTAAAACTGTATGCAATGTTTCTGACTCTCGTTTTTTTGGTCGAACTGGTCGCTGCCATCGTAGGATTTGTTTTCAGACATGAGATTAAGAACAGCTTTAAGAATAATTATGAGAAGGCTTTGAAGCAGTATAACTCTACAGGAGATTATAGAAGCCATGCAGTAGACAAGATCCAAAATACGTTGCATTGTTGTGGTGTCACCGATTATAGAGATTGGACAGATACTAATTATTACTCAGAAAAAGGATTTCCTAAGAGTTGCTGTAAACTTGAAGATTGTACTCCACAGAGAGATGCAGACAAAGTAAACAATGAAGGTTGTTTTATAAAGGTGATGACCATTATAGAGTCAGAAATGGGAGTCGTTGCAGGAATTTCCTTTGGAGTTGCTTGCTTCCAACTGATTGGAATCTTTCTCGCCTACTGCCTCTCTCGTGCCATAACAAATAACCAGTATGAGATAGTGTAACCCAATGTATCTGTGGGCCTATTCCTCTCTACCTTTAAGGACATTTAGGGTCCCCCCTGTGAATTAGAAAGTTGCTTGGCTGGAGAACTGACAACACTACTTACTGATAGACCAAAAAACTACACCAGTAGGTTGATTCAATCAAGATGTATGTAGACCTAAAACTACACCAATAGGCTGATTCAATCAAGATCCGTGCTCGCAGTGGGCTGATTCAATCAAGATGTATGTTTGCTATGTTCTAAGTCCACCTTCTATCCCATTCATGTTAGATCGTTGAAACCCTGTATCCCTCTGAAACACTGGAAGAGCTAGTAAATTGTAAATGAAGTAATACTGTGTTCCTCTTGACTGTTATTTTTCTTAGTAGGGGGCCTTTGGAAGGCACTGTGAATTTGCTATTTTGATGTAGTGTTACAAGATGGAAAATTGATTCCTCTGACTTTGCTATTGATGTAGTGTGATAGAAAATTCACCCCTCTGAACTGGCTCCTTCCCAGTCAAGGTTATCTGGTTTGATTGTATAATTTGCACCAAGAAGTTAAAATGTTTTATGACTCTCTGTTCTGCTGACAGGCAGAGAGTCACATTGTGTAATTTAATTTCAGTCAGTCAATAGATGGCATCCCTCATCAGGGTTGCCAGATGGTGATAACAGTGTAAGGCCTTGGGTCTAAGGCATCCACGACTGGAAGGGACTACTGATGTTCTGTGATACATCAGGTTTCAGCACACAACTTACATTTCTTTGCCTCCAAATTGAGGCATTTATTATGATGTTCATACTTTCCCTCTTGTTTGAAAGTTTCTAATTATTAAATGGTGTCGGAATTGTTGTATTTTCCTTAGGAATTCAGTGGAACTTATCTTCATTAAATTTAGCTGGTACCAGGTTGATATGACTTGTCAATATTATGGTCAACTTTAAGTCTTAGTTTTCGTTTGTGCCTTTGATTAATAAGTATAACTCTTATACAATAAATACTGCTTTCCTCTAAAAAGATCGTGTTTAAATTAACTTGTAGAAAATCTGCTGGAATGGTTGTTGTTTTCCACTGAGAAAGCTAAGCCCTACATTTCTATTCAGAGTACTGTTTTTAGATGTGAAATATAAGCCTGCGGCCTTAACTCTGTATTAAAAAAAATGTTTTTGTTTAAAAAAAACTGTTCCCATAGGTGCAGCAAACCACCATGGCACATGTATACCTATGTAACAAACCTGCACATTCTGTATCTATGTAACAAATCGCAGCACAGGAGTCCCCTGGGCTCCCTCAGGCTCTGGTATGACATATTTGAGCCATATAAATTCAGCTTCTCCTCTGGCATCTGTTAGCCGACTCACTTGCAACTCCACCTCAGCAGTGGTCTCTCAGTCCTCTCAAAGCAAGGAAAGAGTACTGTGTGCTGAGAGACCATGGCAAAGAATCCTCCAGAGAATTGTGAAGACTGTCACATTCTAAATGCAGAAGCTTTTAAATCCAAGAAAATATGTAAATCACTTAAGATTTGTGGACTGGTGTTTGGTATCCTGGCCCTAACTCTAATTGTCCTGTTTTGGGGGAGCAAGCACTTCTGGCCGGAGGTACCCAAAAAAGCCTATGACATGGAGCACACTTTCTACAGCAATGGAGAGAAGAAGAAGATTTACATGGAAATTGATCCTGTGACCAGAACTGAAATATTCAGAAGCGGAAATGGCACTGATGAAACATTGGAAGTGCACGACTTTAAAAACGGATACACTGGCATCTACTTCGTGGGTCTTCAAAAATGTTTTATCAAAACTCAGATTAAAGTGATTCCTGAATTTTCTGAACCAGAAGAGGAAATAGATGAGAATGAAGAAATTACCACAACTTTCTTTGAACAGTCAGTGATTTGGGTCCCAGCAGAAAAGCCTATTGAAAACCGAGATTTTCTTAAAAATTCCAAAATTCTGGAGATTTGTGATAACGTGACCATGTATTGGATCAATCCCACTCTAATATCAGTTTCTGAGTTACAAGACTTTGAGGAGGAGGGAGAAGATCTTCACTTTCCTGCCAACGAAAAAAAAGGGATTGAACAAAATGAACAGTGGGTGGTCCCTCAAGTGAAAGTAGAGAAGACCCGTCACGCCAGACAAGCAAGTGAGGAAGAACTTCCAATAAATGACTATACTGAAAATGGAATAGAATTTGATCCCATGCTGGATGAGAGAGGTTATTGTTGTATTTACTGCCGTCGAGGCAACCGCTATTGCCGCCGCGTCTGTGAACCTTTACTAGGCTACTACCCATATCCATACTGCTACCAAGGAGGACGAGTCATCTGTCGTGTC
Genoma Humano: • ~ 60.000 genes• 1,1% exons• 24% introns• 75% intergênico
Projeto Genoma Humano
人和人之间的差别咋就这么大呢 ?
法 DNA 分型简史( Brief History of Forensic DNA Typing )
• 1980 - Ray White 第一次使用限制性片段长度多态标记 (RFLP marker)
• 1985 - Alec Jeffreys 发现多位点可变数目重复探针 ( VNTR probes )
• 1985 – PCR 的第一篇文章发表• 1988 – FBI 开始使用 DNA 进行法科学调查• 1991 –STR 的第一篇文章发表• 1995 - FSS 建立 DNA 数据库• 1998 - FBI 启动 CODIS 数据库
法 DNA 技术的具体应用(一)法庭案件( Forensic cases ) -- matching suspect with evidence
DNA 在案件中的应用 ( DNA Use in Forensic Cases )
• 多数是强奸案 (>2 out of 3)• 在物证和嫌疑犯间寻找匹配• 必须和受害人的 DNA 数据相比对
•混合斑( Mixtures )•DNA 降解 ( DNA degraded)
•PCR 抑制剂的存在( Inhibitors to PCR )
Challenges
A Need To
微量DNA 检材
烟头邮票……
毛发指纹
法医 DNA 检测的特点( 1 )•犯罪现场可用的生物样本很少是“干净的” ( Crime Scene samples rarely “clean” )
–污垢( Dirt ) - 表面( Surface )–阳光( Sunlight ) - 热( Heat )–潮湿( Moisture ) - 时间( Time )–细菌( Bacteria ) - 场所( Location )–化学品( Chemicals ) - 核酸酶 ( Nucleases )
•DNA 样品的数量常常是有限的( Amount of DNA is often limited )
–必须妥善保存样品( Must conserve sample )–留取足够的样品以便再次分析( Save for possible
defense re-analysis )–每次分析使用足够的样品( Use enough to get
good results )
法医 DNA 检测的特点( 2 )
•亲子鉴定( Paternity testing ) -- identifying father
法 DNA 技术的具体应用(二)
Modern Use of Y-STR TestingCaptured 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)
历史调查( Historical investigations )
法 DNA 技术的具体应用(三)
Thomas Jefferson II
Field Jefferson Peter Jefferson
President Thomas Jefferson
Eston Hemings Thomas Woodson
Different Y Haplotype
Same Y Haplotype
Jefferson Y Haplotype
Jefferson Y Haplotype
?
Historical Investigation DNA Study
走
出
非
洲
http://www.nyfd.com/
失踪人员调查( Missing persons investigations )群难事故( Mass disasters ) -- putting pieces back together
Time (Minutes)7654321
Abs
orba
nce
(mV
)
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
-1
法 DNA 技术的具体应用(四)
犯罪 DNA 数据库( Convicted felon DNA database
s )
法 DNA 技术的具体应用(五)
DNA 证据的来源( Sources of DNA Evidence )
• 血液 (Blood)• 精液 (Semen)• 唾液 (Saliva)• 尿液 (Urine)• 毛发 (Hair)• 牙齿 (Teeth)• 骨骼 (Bone)• 组织 (Tissue)
从犯罪现场或亲权鉴定中收集样品 生物学
DNA 提取DNA 提取 DNA定量
DNA定量
PCR 扩增多个 STR 遗传标记
PCR 扩增多个 STR 遗传标记
技术分离和检查 PCR 产物
(STR 等位基因 )样品所具有的
基因型
遗传学将样品的基因型和其它样
品的基因型相比较将样品的基因型和其它样
品的基因型相比较
如相互吻合,将基因型资料与群体数据库相比对
如相互吻合,将基因型资料与群体数据库相比对
计算随机匹配概率计算随机匹配概率
DNA 样品处理的步骤( Steps in DNA Sample Processing )
引物延伸Make copies
(extend primers)
初始模板Starting DNA
Template
5’
5’
3’
3’
5’
5’
3’
3’
添加引物Add primers
(anneal)
5’3’
3’5’
前引物Forward primer
后引物Reverse primer
聚合酶链反应Polymerase Chain Reaction (PCR)
变性为单链Separate strands
(denature)
5’
5’3’
3’
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
目的片段随循环次数指数级增加PCR Copies DNA Exponentially through
Multiple Thermal Cycles
起始目的片段( Original DNA target region )
Thermal cycleThermal cycleThermal cycle
诺贝尔奖( The Nobel Laureate )
• Invented by Kary Mullis in 1983.• First published account appeared in 1985.• Awarded Nobel Prize for Chemistry in
1993.
短串联重复Short Tandem Repeats (STRs)
不同样本间引物结合区相同而基序重复次数是可变的( the repeat region is variable between samples while the flanking regions where PCR primers bind are constant )
7 repeats
8 repeats
AATG
杂合体( Homozygote ) = both alleles are the same length
纯合体( Heterozygote ) = alleles differ and can be resolved from one another
170 bp170 bp195 bp195 bp
TCAT repeat unitTCAT repeat unit
DNA 检测的具体步骤
DNA 提取( DNA collected )1
2
3
4
DNA 扩增(DNA amplified if necessary)
DNA 目的片段(DNA cut into fragments)
DNA 片段比较(fragments compared)
犯罪现场 嫌疑人 1 嫌疑人 2
犯罪现场样品和参照样品( Crime Scene Samples & Reference Samples )
提取和纯化 DNA (Extract and Purify DNA)
添加引物和其它试剂(Add primers and other reagents)
PCR 扩增( PCR Amplification )
• 扩增目的片段( DNA regions flanked by
primers are amplified )
ABI310 遗传分析仪 ( 310 Genetic Analyzer )
毛细管
聚合胶注射器
自动取样盘缓冲液
电极
缓冲液
310 遗传分析仪原理ABI 310 Genetic Analyzer: Capillary Electrophoresis
检测窗(DetectorWindow)
毛细管电泳( Capillary Electrophoresis )
Figure 14.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
310 分析原始图谱 ( Raw data )
矩阵处理
(Matix)
D3S1358(8 alleles)
VWA(14 alleles)
D16S539(9 alleles)
D2S1338(14 alleles)
Blue panel
Green panel
Yellow panel
Orange panel
D21S11(24 alleles)
D8S1179(12 alleles)
D18S51(23 alleles)
TH01(10 alleles)
FGA low(19 alleles)
FGA high(9 alleles)
250 bp*139bp 200 bp160 bp300 bp 340 bp 350 bp
150 bp
LIZ-labeled GS500 DNA sizing standard
100 bp
Red panel
D19S433(15 alleles)
D5S818(10 alleles)
TPOX(8 alleles)
D13S317(8 alleles)
D7S820(10 alleles)
CSF1PO(10 alleles)
AMEL(2 alleles)
Figure 5.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
通过与等位基因标准比对来进行 STR 分型 ( STR genotyping is performed by comparison of sample data to allelic ladders )
Microvariant allele
统计分析
0.222 x 0.222 x 2
= 0.1
= 0.1
1 in 79,531,528,960,000,000
1 in 8 万万亿( quadrillion )
1 in 10 1 in 111 1 in 20
1 in 22,200
x x
1 in 100 1 in 14 1 in 81
1 in 113,400
x x
1 in 116 1 in 17 1 in 16
1 in 31,552
x x
统计分析
DNA 指纹
(DNA fingerprint)
一例强奸案的 DNA 图谱
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…
D8S1179
D21S11
D7S820
CSF1PO
D19S433
vWA TPOX D18S51
D3S1358
父 10-11 32.2 8-11 10-10 14-15 14-16 11-12 17-17 15-15
母 14-15 29-29 10-12 10-11 13-14 18-19 11-12 13-15 16-17
子 11-14 29-32.2 8-10 10-10 14-14 14-18 12-12 13-17 16-17
TH01 D13S317
D16S539
D2S1338
D5S818
FGA D1S2142
D15S659
D14S306
父 9-9 8-12 9-11 19-22 10-12 22.2-23
13-16 9-14 12-12
母 9-9 9-12 9-11 18-23 11-12 22-24 16 16 14 15 13 13
子 9-9 8-12 9-9 22-23 12-12 22-22.2
16 16 14 15 12 13
D13S1492
D18S865
D12S391
D20S161
D3S1754
D4S2366
D12S375
D1S549
Amel
父 20-20 10-10 8-11 17-18 11-13 9-14 15-17 12-16 X Y
母 14-14 10-11 6-7 16-17 13-14 11-13 15-15 15-16 X X
子 14-20 10-11 7-11 16-17 11-13 11-14 15-17 12-15 X Y
一例亲子鉴定的 STR 分型结果
Applied Biosystems PRISM 377(Gel, 34-96 lanes)
Applied Biosystems PRISM 3100(Capillary, 16 capillaries)
Applied Biosystems PRISM 3700(Capillary, 96 capillaries)
DNA 数据库
( DNA Database )
CODIS DNA 数据库 ( FBI’s CODIS DNA Database )
联合 DNA 索引系统( Combined DNA Index System )• Used for linking serial crimes and unsolved
cases with repeat offenders• Launched October 1998• Links all 50 states• Requires >4 RFLP markers
and/or 13 core STR markers• Current backlog of >1,000,000 samples
13 个 CODIS 核心 STR 位点及其染色体定位 Core STR Loci with Chromosomal Positions
CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539 D18S51
D8S1179
D3S1358
FGA
VWA
AMEL
AMEL
DNA in database does not only convict but also free individuals:
Case example from New York City
A woman was attacked in her building. She was injured and
bleeding heavily. A neighbor saw a man flee from the scene. The
neighbor identified another tenant as the attacker. This man
“confessed” to the crime.
On the stairway leading away from the scene were a few drops of blood.
The blood on the stairs was of male origin but did not match the arrested suspect.
The detectives were not sure if the droplets were actually connected to the assault.
BUT…..
The profile generated a local CODIS hit
• The DNA profile from the blood droplets on the stairs was consistent with a semen donor from a second rape in the Bronx.
• The new suspect was arrested and the former defendant was exonerated from the crime that he did not commit.
• Eye witness testimony had been unreliable.
英国国家 DNA 数据库The National DNA Database
• 1995 年 4 月建立警察智能 DNA 数据库 • 2004 年 6 月 30 日 :
– 2,396,429 份罪犯数据 – 227,010 份未匹配的犯罪现场 DNA 数据
• 2004 年 6 月成功匹配 3,409 例案件的 DNA 数据• 2001 年 5 月 ~ 2004 年 6 月成功匹配 134,965 例案件
的 DNA 数据• 2004-2007: 整个欧洲的 DNA 数据库共享
我国的 DNA 数据库建设
• 失踪儿童数据库• 犯罪嫌疑人数据库
Yesterday Nowadays Tomorrow
法 DNA 技术的前景和展望
结 束 语
随着 DNA 技术迅速的普及和发展,不法分子如果还想以身试法的话,他可真的要三思而后行了
Thanks for your attention.
Questions?