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 –The Combined DNA Index System (CODIS) –DNA paternity testing –Mitochondrial DNA testing

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.

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.

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

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

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

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

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.

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.

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.

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.

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

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.

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

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.

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.

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.

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

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.

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

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

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

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.

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.

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

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.

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.

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

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.

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

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.

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

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.

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.

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.

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.