生化基础法医学

1、会计学1生化基础法医学生化基础法医学4核酸5端可以被标记：标记物(放射性、非放射性)，标记要点，显示方法的差异。 5限制性核酸内切酶（识别特异性序列）、特点、意义。 6遗传变异的分子基础：突变、重组、转座子、(精子或卵子 成熟 过程)的交换重组 DNARNA转录、剪接、重排 蛋白质翻译调控反转录逆转录病毒反义治疗抗生素化疗放疗化疗放疗核酶microRNA复制蛋白质构像的改变-疯牛病（月元）中心法则 DNA标记的优点1. 多态性高：DNA指纹(DNA fingerprint)，5000 2万个STR位点，1个SNP位点/1kb，MVR-minsatellite; 单个STR位点的等位基因数量多；

2、 个体分辨率(discriminating power)高2. 没有两个人(单卵双生除外)的DNA序列是完全相同的;3. 干燥后可以长期保存（9400 y）;4. 线粒体DNA多拷贝（几个1000个），检测灵敏度高;5. DNA分析技术成熟，可机械化、自动化.细胞核DNA：常染色体DNA性染色体DNA线粒体DNA人 类 DNA 分 类 RBC(膜抗原及其中的酶) 基因(倾向保守) HLA 染色体DNA 编码 外显子 血清蛋白 (父母源) 调控区，内含子 同工酶 单拷贝(spacer) 卫星DNA(着丝粒周围) 串联重复 小卫星DNA(散在) 非编码 (VNTR10%) 微卫星DNA(STR)D

3、NA 多拷贝 SINES(短) 分类 (重复序列) 散在重复 50000bp L1 线粒体DNA L2HS (母源) KpnI (父:10-4) 非编码区 D-LOOP(非重复序列)the repeat region is variable between samples while the flanking regions where PCR primers bind are constant7 repeats8 repeatsAATGHomozygote = both alleles are the same lengthHeterozygote = alleles differ and

4、can be resolved from one anotherPowerPlex 16 LaddersD3S1358D16S539D21S11D18S51Penta DD13S317CSF1POD7S820Penta ETH01D5S818VWAD8S1179TPOXAMELFGA(9)(9)(25)(22)(20)(10)(9) (9)(9)(10)(14)(13)(12)(8)(27)13 CODIS Core STR Loci with Chromosomal PositionsCSF1POD5S818D21S11TH01TPOXD13S317D7S820D16S539D18S51D8

5、S1179D3S1358FGAVWAAMELAMEL遗传标记举例the repeat region is variable between samples while the flanking regions where PCR primers bind are constant7 repeats8 repeatsAATGHomozygote = both alleles are the same lengthHeterozygote = alleles differ and can be resolved from one another170 bp195 bpDifferent prime

6、r sets produce different PCR product sizes for the same STR alleleTCAT repeat unitPowerPlex 16 LaddersD3S1358D16S539D21S11D18S51Penta DD13S317CSF1POD7S820Penta ETH01D5S818VWAD8S1179TPOXAMELFGA(9)(9)(25)(22)(20)(10)(9) (9)(9)(10)(14)(13)(12)(8)(27)D8S1179D21S11D7S820CSF1POD3S1358TH01D13S317D16S539D2S

7、1338D19S433D18S51TPOXVWAAMELD5S818FGAGS500 LIZ size standardMarker Name GenBank Accession Repeat Motif Allele Range PCR Product Sizes Reference DYS19 X77751 TAGA 8-16 178-210 bp Roewer 1992 DYS385 Z93950 GAAA 10-22 252-300 bp Schneider 1998 DYS388 G09695 ATT 12-17 128-143 bp Kayser 1997 DYS389 I DYS

8、389 II G09600 G09600 (TCTG) (TCTA) (TCTG) (TCTA) I: 7-13 II:23-31 239-263 bp 353-385 bp Kayser 1997 Kayser 1997 DYS390 G09611 (TCTA) (TCTG) 18-27 191-227 bp Kayser 1997 DYS391 G09613 TCTA 8-13 275-295 bp Kayser 1997 DYS392 G09867 TAT 7-16 236-263 bp Kayser 1997 DYS393 G09601 AGAT 9-15 108-132 bp Kay

9、ser 1997 YCAIII AC006370 CA 19-25 192-204 bp Kayser 1997 DYS434 AC002992 ATCT 8-11 110-122 bp Ayub 2000 DYS435 AC002992 TGGA 9-13 210-228 bp Ayub 2000 DYS436 AC005820 GTT 10-15 128-143 bp Ayub 2000 DYS437 AC002992 TCTA 8-11 186-202 bp Ayub 2000 DYS438 AC002531 TTTTC 6-12 203-233 bp Ayub 2000 DYS439

10、AC002992 AGAT 9-14 238-258 bp Ayub 2000 Y-GATA-A4 G42670 AGAT 11-14 242-254 bp White 1999 Y-GATA-A7.1 G42675 ATAG 7-12 161-181 bp White 1999 Y-GATA-A7.2 G42671 TAGA 8-12 174-190 bp White 1999 Y-GATA-A8 G42672 TCTA 8-14 219-244 bp White 1999 Y-GATA-A10 G42674 TATC 11-14 160-172 bp White 1999 Y-GATA-C

11、4 G42673 TATC 11-16 251-271 bp White 1999 Y-GATA-H4 G42676 TAGA 10-13 362-370 bp White 1999 Y-STR MarkersJ.M. Butler, Forensic DNA Typing, Table 8.1 序列多态性 D-loop区域 长度多态性 nt514-nt523：CA重复 C-stretch: polyC 9bp DelitionmtDNA序列多态性mtDNA 序列的表示方法（例：Andersons sequence）序号nt序号nt序号nt序号nt序号nt Steps in DNA Sampl

12、e ProcessingBiologyDNAExtractionDNAQuantitationPCR Amplificationof Multiple STR markersTechnologySeparation and Detection of PCR Products(STR Alleles)Sample Genotype DeterminationGeneticsComparison of Sample Genotype to Other Sample ResultsIf match occurs, comparison of DNA profile to population dat

13、abasesGeneration of Case Report with Probability of Random MatchSample Obtained from Crime Scene or Paternity InvestigationAllelepossibilitiesSampleGenotype1 in 181 locus:1 in 181 in 50X3 loci:1 in 180001 in 20X2 loci:1 in 360Hypothetical likelihood of occurrence1 in 44X4 loci:1 in 7920009 loci: 1 i

14、n 101016 loci: 1 in 1017Current World Pop: 6.3 billionPowerPlex 16 SystemProduct Overview5 bpAdvantages of Pentas vs. Tetranucleotides:Less Stutter artifactsHighly discriminating lociGreater separation of individual allelesFewer MicrovariantsPentanucleotidesHomozygote = both alleles are the same len

15、gthHeterozygote = alleles differ and can be resolved from one anotherShort Tandem Repeats (STRs)Repeat region is variable (polymorphic) Each variant is referred to as an alleleFlanking region is constantKEY: Alleles are distinguished by length7 repeats8 repeatsAATG100 bp500 bp200 bp300 bp400 bp600 b

16、pTH01D3D21D18Penta ED5D13D7D16CSFPenta DAvWAD8TPOXFGA100500200300400600Allele CallsChallenges辐射：-，-，-射线，X-ray，中子，质子及紫外线化学诱变：乙酰亚胺（ethylenimine）及其衍生物，杀虫剂， 石油工业产品，诱染色体畸变三乙硫代磷酰胺（thiotepa） 染料，防火，防水，染色体畸变 芥子气（mustard gas）; 塑料业，畸胎，致癌，诱变; 亚硝酸（nitrosamine）, 烟草中，致癌、诱变; 乙二胺四乙酸（EDTA）, Captan 杀真菌剂, 致畸胎; 链霉黑素（str

17、eptonigrin）抗生素、癌化疗剂，致癌、诱变 AF-（呋喃糖酰胺 furylfuramide） 食品添加剂：（豆腐、火腿、香肠中的）致癌、诱变、染色体畸变 试剂：丙稀酰胺、联苯胺、溴乙锭等 DNA复制时出现的差错：常见于线粒体的异质性DNA in the CellTarget Region for PCRchromosomecell nucleusDouble stranded DNA moleculeIndividual nucleotidesMake copies (extend primers)Starting DNA Template55335533Add primers (an

18、neal)5335Forward primerReverse primerDNA Amplification with the Polymerase Chain Reaction (PCR)Separate strands (denature)5533In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are createdPCR Copies DNA Exponentially through Multiple Thermal CyclesOriginal DNA target regionT

19、hermal cycleThermal cycleThermal cycle两份两份DNA样本的比对结果样本的比对结果HVI A1B1 57.9CForensicsTime (Minutes)7654321Absorbance (mV)11109876543210 4.39 4.70 5.02+ Reference未知样本一+ Refernce未知样本二Sample alone with reference 1 with reference 2 with reference 3Sample 1 mtDNA profile A1B2Sample 2 mtDNA profile A1B2Time

20、(Minutes)7654321Absorbance (mV)87654321Time (Minutes)7654321Absorbance (mV)87654321Time (Minutes)7654321Absorbance (mV)14131211109876543210Time (Minutes)7654321Absorbance (mV)121110987654321Time (Minutes)7654321Absorbance (mV)109876543210Time (Minutes)7654321Absorbance (mV)1514131211109876543210Time (Minutes)7654321Absorbance (mV)6543210Time (Minutes)7654321Absorbance (mV)6543210Profile of samples mixed with references DNA标记的优点1. 多态性高：DNA指纹(DNA fingerprint)，5000 2万个STR位点，1个SNP位点/1kb，MVR-minsatellite; 单个STR位点的等位基因数量多； 个体分辨率(discriminating power)高2. 没有两个人(单卵双生除外)的DNA