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Chapter 14

Chapter 14. Forensic DNA Typing. Objectives. Students should gain an understanding of: The use of the polymerase chain reaction (PCR) to make many copies of a DNA sequence Short tandem repeats (STRs) and their forensic importance The use of electrophoresis to analyze STRs

vivian-wilkerson
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Chapter 14

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  1. Chapter 14 Forensic DNA Typing

  2. Objectives • Students should gain an understanding of: • The use of the polymerase chain reaction (PCR) to make many copies of a DNA sequence • Short tandem repeats (STRs) and their forensic importance • The use of electrophoresis to analyze STRs • The Combined DNA Index System (CODIS) • DNA paternity testing • Mitochondrial DNA testing

  3. Introduction • The DNA in all cells of an individual is the same through the body. • DNA contains repeated sequences of genetic codes with core sequences that are unique to particular individuals. • The genetic code can be determined from a small amount of DNA.

  4. Restriction Fragment Length Polymorphisms (1 of 3) • DNA contains genes that control production of proteins in the body. • Other sections act as spacers between the coding areas. • The sequences of bases in the noncoding regions are used for DNA profiling. • The sequences vary greatly from person to person.

  5. Restriction Fragment Length Polymorphisms (2 of 3) • RFLP allows for the individualization of DNA evidence • Step 1: DNA is extracted from a chromosome • Step 2: restriction enzymes cut the DNA strands into fragments at specific base sequences

  6. Restriction Fragment Length Polymorphisms (3 of 3) • Disadvantages of RFLP • Takes 6–8 weeks to obtain results • Requires a large sample of intact, nondegraded DNA • Not amenable to high-volume sample processing • Produces very large DNA strands that are often damaged when the recovered DNA is partially degraded

  7. Polymerase Chain Reaction: A DNA Copy Machine (1 of 3) • Advantages of PCR • Allows many copies of a portion of DNA sequence to be manufactured in the DNA lab • Amplifies only those DNA regions that are of interest • Is fast and extremely sensitive

  8. Polymerase Chain Reaction: A DNA Copy Machine (2 of 3) • Thermocycling: DNA is repeatedly heated and cooled • 194 °F: the two complementary DNA strands separate • 140 °F: each primer finds and binds to its complementary sequence on the DNA strand • 162 °F: the DNA polymerase enzyme adds bases to extend the primer and to build a DNA strand that is complementary to the sample DNA

  9. Polymerase Chain Reaction: A DNA Copy Machine (3 of 3) • The thermocycling process is repeated to make more copies. • Each heating–cooling cycle doubles the number of copies. • DNA laboratory and technicians must take extreme care to eliminate extraneous DNA from the PCR amplification area.

  10. Short Tandem Repeats • STRs: locations on the sample DNA that contain a short sequence of bases that is repeated over and over • Four-base repeats are typically used for forensic purposes. • STRs are often recovered from bodies or stains that have started to decompose. • They can be amplified very quickly.

  11. DNA Sequence Variations among Individuals • Individuals differ genetically because they possess different combinations of alleles at numerous locations in their genomes. • Only 3% of a person’s DNA is involved in coding for proteins. • Mutations in noncoding regions have no effect on the phenotype of a person. • Loci selected for DNA typing are selectively neutral; they confer neither benefit nor harm to the individual’s ability to reproduce.

  12. Inheritance of Alleles • Alleles are inherited from an individual’s parents following the fundamental rules of genetics. • Different individuals posses different alleles in numerous loci in their genomes. • Investigators measure the length of STRs at different locations to determine a person’s genetic identity.

  13. Analyzing the STR by Electrophoresis (1 of 3) • Electrophoresis • Causes ions in solution to migrate under the influence of an electric field • Separates STRs according to their length: smaller DNA molecules move faster • Establishes the number of repeats and elucidates the genotype of the individual at each amplified locus

  14. Analyzing the STR by Electrophoresis (2 of 3) • Gel electrophoresis • After voltage is applied 2–3 hours, electrophoresis stops and the DNA is made visible. • Each group of similar-length molecules appears as a narrow band in the gel. • By comparing the locations of the bands in each sample lane to the ladder, the technician can determine the STR type for each sample. • Gel electrophoresis is slow, is difficult to automate, and can be dangerous.

  15. Analyzing the STR by Electrophoresis (3 of 3) • Capillary electrophoresis • Allows for greater heating than is possible with a slab gel • Uses a higher voltage, so molecules migrate much faster • Produces high-speed, high-resolution separations on extremely small samples • Uses laser fluorescence: fluorescent dye is attached to the PCR primer that amplifies the STR region of interest

  16. Multiplex DNA Analysis (1 of 4) • Multiple STR loci may undergo PCR amplification and be analyzed simultaneously. • Multiplexing is accomplished by placing each of the four dyes on specific primers and by adjusting the size of the STR amplicon produced.

  17. Multiplex DNA Analysis (2 of 4) • Multiplexing by size • Amplicons from different loci that are different sizes are clearly separated from one another and appear at different locations on the x-axis in the CE analysis. • It isn’t possible to multiplex more than five or six loci.

  18. Multiplex DNA Analysis (3 of 4) • Multiplexing by dye color • Different dyes amplify STR loci that are the same size and cannot be separated using CE.

  19. Multiplex DNA Analysis (4 of 4) • Multiplexing with multiple capillaries: capillary array electrophoresis • Parallel capillaries lie next to one another and process multiple samples simultaneously • Technique reduces the time between when a DNA sample is collected at the crime scene and when it is actually analyzed

  20. Forensic STRs • Most DNA databases rely on 10 or more STR loci, each of which is found on a different chromosome. • Standard nomenclature is used to designate the location of a DNA marker. • If the marker is part of a gene or falls within it, the gene name is used. • If the STR falls outside a gene region, its name indicates the chromosome and locus on which it is found.

  21. CODIS (1 of 4) • Combined DNA Index System • Created in 1994 as part of the DNA Identification Act • Consists of a national database containing the DNA of individuals convicted of sexual and violent crimes • Assisted in 20,000 investigations in 2004

  22. CODIS (2 of 4) • Three tiers of CODIS • Local: labs maintain a local DNA index • State: combines the profiles of all local labs • National: compares profiles of all state systems

  23. CODIS (3 of 4) • All 50 states maintain databases for sexual offenders and convicted murderers • 49 states include violent felons • 43 states include all felons

  24. CODIS (4 of 4) • Use of CODIS • The computer compares the DNA profile submitted with profiles on file in the network. • If a match is found in the Convicted Offender Index, the lab is sent the identity of the perpetrator. • If a match is found in the Forensic Index, two crimes have been linked together. The labs must then verify the match; law enforcement may then pool resources to solve the crimes.

  25. Interpretation of DNA Profiles (1 of 3) • It is easier to use DNA to exclude a person from suspicion than to prove that the person is the only suspect. • The Innocence Project reports that three times more suspects are proven innocent by DNA analysis than are proven guilty. • The loci used for DNA matches must be chosen to minimize the chance that two people will have the same profile.

  26. Interpretation of DNA Profiles (2 of 3) • Hardy–Weinberg principle: allele frequencies remain constant from generation to generation and allele frequencies can be easily calculated • Frequency of a particular homozygote = allele frequency squared • Expected frequency for a heterozygote = 2 × the product of the two allele frequencies

  27. Interpretation of DNA Profiles (3 of 3) • Interpreting multiple DNA profiles: • Prevalence of a particular CODIS profile in the general population: multiply the genotype frequencies for all the loci together • Likelihood ratio: compares the probabilities of alternative events • The true discriminating power of CODIS is achieved by multiplying the individual frequencies of the 13 loci.

  28. Paternity Testing (1 of 2) • A child can receive only one of the father’s alleles and one of the mother’s alleles. • A familial pattern should be obvious by comparing the DNA profiles of mother, father, and child.

  29. Paternity Testing (2 of 2) • Paternity index: the likelihood that an allele from the child supports the assumption that the tested man is the true biological father • Combined paternity index: determined by multiplying the individual PIs for each locus tested

  30. Mitochondrial DNA Analysis (1 of 5) • Examination of recovered mitochondrial DNA is useful in circumstances of badly decomposed or burned bodies, old bones, and human hair without follicular tags • mtDNA is rarely used in criminal proceedings. • It has been useful for historical investigations.

  31. Mitochondrial DNA Analysis (2 of 5) • Mitochondrial DNA (mtDNA) • Is a circular DNA molecule that is only 16,569 pairs in circumference • Has no noncoding elements; every base has a function • For the most part, is the same in all individuals

  32. Mitochondrial DNA Analysis (3 of 5) • Variations in mtDNA • The D-loop contains two hypervariable regions whose sequences vary. • A difference of less than 3% is expected between unrelated individuals.

  33. Mitochondrial DNA Analysis (4 of 5) • DNA sequencing: determines the sequence of bases along a DNA strand • Anderson sequence: the first mtDNA hypervariable sequence to be determined; serves as a reference sample

  34. Mitochondrial DNA Analysis (5 of 5) • mtDNA is inherited from a person’s mother. • All brothers and sisters of the same mother have the same mtDNA, which is also the same as the mtDNA of their maternal grandmother and their mother’s siblings.

  35. The Y Chromosome: STRs and SNPs (1 of 2) • STR analysis is directed at the Y chromosome. • The Y chromosome contains polymorphisms that might eventually be used as forensic markers.

  36. The Y Chromosome: STRs and SNPs (2 of 2) • Single-nucleotide polymorphisms (SNPs) (2 of 2) • The base difference occurs at only one specific site. • SNPs at different loci can be determined simultaneously, producing an SNP DNA profile.

  37. Low-Copy-Number DNA Typing • Applications • Used when the quantifying test indicates too little DNA is available to perform a regular DNA analysis • Used only in cases where standard typing protocols have already failed • As the amount of suspect DNA decreases, the chance of contamination by DNA from other sources increases.

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