【正文】 Make copies (extend primers) Starting DNA Template 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ Add primers (anneal) 5’ 3’ 3’ 5’ Forward primer Reverse primer DNA Amplification with the Polymerase Chain Reaction (PCR) Separate strands (denature) 5’ 5’ 3’ 3’ In 32 cycles at 100% efficiency, billion copies of targeted DNA region are created PCR Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target region Thermal cycle Hypothetical pedigree indicating some of the relatives of a person who could be used for identification 線粒體 DNA分析在法醫(yī)學(xué)上的應(yīng)用 單純樣本的圖形為單峰 對于混合樣本，如為同一人，則圖形也為單峰 。 塑料業(yè) ， 畸胎 ， 致癌 ， 誘變 。 Steps in DNA Sample Processing Biology DNA Extraction DNA Quantitation PCR Amplification of Multiple STR markers Technology Separation and Detection of PCR Products (STR Alleles) Sample Genotype Determination Geics Comparison of Sample Genotype to Other Sample Results If match occurs, parison of DNA profile to population databases Generation of Case Report with Probability of Random Match Sample Obtained from Crime Scene or Paternity Investigation Power of Discrimination Through Multiplexing Allele possibilities Sample Genotype 1 in 18 1 locus: 1 in 18 1 in 50 X 3 loci: 1 in 18000 1 in 20 X 2 loci: 1 in 360 Hypothetical likelihood of occurrence 1 in 44 X 4 loci: 1 in 792022 9 loci: ~1 in 1010 16 loci: ~1 in 1017 Current World Pop: ~ billion 結(jié)束 Commonly Used Terms ? Allele: An alternative form of a gene. ? Allele Frequency: The proportion of a particular allele among the chromosomes carried by individuals in a population. ? Homozygosity: The presence of the same alleles at one or more loci on homologous chromosomes. ? Heterozygosity: The presence of different alleles at one or more loci on homologous chromosomes. ? Locus (pl. Loci): The position of a gene or chromosome segment on a chromosome. Alleles are located at identical loci on homologous chromosomes. ? Mixture: The presence of two or more contributors to a given sample. ? Multiplexing: A PCR approach that amplifies several alleles simultaneously, greatly increasing throughput. ? Short tandem repeats (STR): Multiple copies of an identical DNA sequence arranged in direct succession in a particular region of a chromosome. PowerPlex 16 System Product Overview 5 bp Advantages of Pentas vs. Tetranucleotides: ?Less Stutter artifacts ?Highly discriminating loci ?Greater separation of individual alleles ?Fewer Microvariants ?Pentanucleotides Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another Short Tandem Repeats (STRs) ? Repeat region is variable (polymorphic) – Each variant is referred to as an allele ? Flanking region is constant KEY: Alleles are distinguished by length 7 repeats 8 repeats AATG ~ ~ ~ ~ Current Forensic STR Multiplexes PowerPlex 16 and PowerPlex 16 BIO 100 bp 500 bp 200 bp 300 bp 400 bp 600 bp TH01 D3 D21 D18 Penta E D5 D13 D7 D16 CSF Penta D A vWA D8 TPOX FGA 100 500 200 300 400 600 Allele Calls Human Identity Testing Applications ? Forensic cases: matching suspect with evidence ? Paternity testing: identifying father ? Convicted felon DNA databases ? Missing persons investigations ? Mass disasters putting pieces back together ? Historical investigations ? Immigration ? Military DNA “dog tag” DNA Use in Forensic Cases ? Most are rape cases (2 out of 3) ? Looking for matches between evidence, victim, and suspect ? Must pare DNA profiles ?Mixtures must be resolved if present ?DNA is often degraded ?Inhibitors to PCR and sample contamination are often present Challenges Overview of PowerPlex 16 System ? Single Amplification Systems ? 13 CODIS loci, 2 penta loci, Amelogenin ? PowerPlex174。 變異 (variation)是絕對的 ：生物進(jìn)化的前提，生物 多樣性 (biovariety) （ DNA多態(tài)性）的成因。 16 Ladders D3S1358