生化基礎(chǔ)法醫(yī)學(xué)

1、會(huì)計(jì)學(xué)1生化基礎(chǔ)法醫(yī)學(xué)生化基礎(chǔ)法醫(yī)學(xué)4核酸5端可以被標(biāo)記：標(biāo)記物(放射性、非放射性)，標(biāo)記要點(diǎn)，顯示方法的差異。 5限制性核酸內(nèi)切酶（識(shí)別特異性序列）、特點(diǎn)、意義。 6遺傳變異的分子基礎(chǔ)：突變、重組、轉(zhuǎn)座子、(精子或卵子 成熟 過程)的交換重組 DNARNA轉(zhuǎn)錄、剪接、重排 蛋白質(zhì)翻譯調(diào)控反轉(zhuǎn)錄逆轉(zhuǎn)錄病毒反義治療抗生素化療放療化療放療核酶microRNA復(fù)制蛋白質(zhì)構(gòu)像的改變-瘋牛?。ㄔ略┲行姆▌t DNA標(biāo)記的優(yōu)點(diǎn)1. 多態(tài)性高：DNA指紋(DNA fingerprint)，5000 2萬個(gè)STR位點(diǎn)，1個(gè)SNP位點(diǎn)/1kb，MVR-minsatellite; 單個(gè)STR位點(diǎn)的等位基因數(shù)量多；

2、 個(gè)體分辨率(discriminating power)高2. 沒有兩個(gè)人(單卵雙生除外)的DNA序列是完全相同的;3. 干燥后可以長(zhǎng)期保存（9400 y）;4. 線粒體DNA多拷貝（幾個(gè)1000個(gè)），檢測(cè)靈敏度高;5. DNA分析技術(shù)成熟，可機(jī)械化、自動(dòng)化.細(xì)胞核DNA：常染色體DNA性染色體DNA線粒體DNA人 類 DNA 分 類 RBC(膜抗原及其中的酶) 基因(傾向保守) HLA 染色體DNA 編碼 外顯子 血清蛋白 (父母源) 調(diào)控區(qū)，內(nèi)含子 同工酶 單拷貝(spacer) 衛(wèi)星DNA(著絲粒周圍) 串聯(lián)重復(fù) 小衛(wèi)星DNA(散在) 非編碼 (VNTR10%) 微衛(wèi)星DNA(STR)D

3、NA 多拷貝 SINES(短) 分類 (重復(fù)序列) 散在重復(fù) 50000bp L1 線粒體DNA L2HS (母源) KpnI (父:10-4) 非編碼區(qū) D-LOOP(非重復(fù)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遺傳標(biāo)記舉例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 序列多態(tài)性 D-loop區(qū)域 長(zhǎng)度多態(tài)性 nt514-nt523：CA重復(fù) C-stretch: polyC 9bp DelitionmtDNA序列多態(tài)性mtDNA 序列的表示方法（例：Andersons sequence）序號(hào)nt序號(hào)nt序號(hào)nt序號(hào)nt序號(hào)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輻射：-，-，-射線，X-ray，中子，質(zhì)子及紫外線化學(xué)誘變：乙酰亞胺（ethylenimine）及其衍生物，殺蟲劑， 石油工業(yè)產(chǎn)品，誘染色體畸變?nèi)伊虼柞０罚╰hiotepa） 染料，防火，防水，染色體畸變 芥子氣（mustard gas）; 塑料業(yè)，畸胎，致癌，誘變; 亞硝酸（nitrosamine）, 煙草中，致癌、誘變; 乙二胺四乙酸（EDTA）, Captan 殺真菌劑, 致畸胎; 鏈霉黑素（str

17、eptonigrin）抗生素、癌化療劑，致癌、誘變 AF-（呋喃糖酰胺 furylfuramide） 食品添加劑：（豆腐、火腿、香腸中的）致癌、誘變、染色體畸變 試劑：丙稀酰胺、聯(lián)苯胺、溴乙錠等 DNA復(fù)制時(shí)出現(xiàn)的差錯(cuò)：常見于線粒體的異質(zhì)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兩份兩份DNA樣本的比對(duì)結(jié)果樣本的比對(duì)結(jié)果HVI A1B1 57.9CForensicsTime (Minutes)7654321Absorbance (mV)11109876543210 4.39 4.70 5.02+ Reference未知樣本一+ Refernce未知樣本二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標(biāo)記的優(yōu)點(diǎn)1. 多態(tài)性高：DNA指紋(DNA fingerprint)，5000 2萬個(gè)STR位點(diǎn)，1個(gè)SNP位點(diǎn)/1kb，MVR-minsatellite; 單個(gè)STR位點(diǎn)的等位基因數(shù)量多； 個(gè)體分辨率(discriminating power)高2. 沒有兩個(gè)人(單卵雙生除外)的DNA