Molecular Biology and Forensic DNA Flashcards

DNA in Cells

  • DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all other organisms. It is located in the cell nucleus and is meticulously folded into chromosome structures to fit within the limited space.

  • There are 22 pairs of autosomal chromosomes (non-sex determining), which are the same in both males and females. These chromosomes contain genes that code for various traits and functions.

  • Sex-determining chromosomes are XX (females) or XY (males). Females have two copies of the X chromosome, while males have one X and one Y chromosome, which determines their sex.

  • One copy of each chromosome is inherited from each parent through sexual reproduction. This ensures genetic diversity and variation in offspring.

  • Of the 3,000,000,000 total base pairs, a small region varies between individuals. These variations, known as polymorphisms, are the basis of genetic differences among individuals.

  • Forensic DNA targets these varying regions to individualize people. By analyzing these specific regions, forensic scientists can create unique DNA profiles for identification purposes.

  • DNA structure:

    • Consists of a phosphate group, a sugar molecule (deoxyribose), and a nitrogenous base (A, T, C, or G). These components form the building blocks of DNA called nucleotides.

    • Has a five prime (5') to three prime (3') direction based on deoxyribose carbon positioning. This directionality is crucial for DNA replication and transcription.

  • Hybridization and double helix structure:

    • Two hydrogen bonds between A and T nucleotides. Adenine (A) always pairs with Thymine (T) through two hydrogen bonds, providing stability to the DNA structure.

    • Three hydrogen bonds between G and C nucleotides. Guanine (G) always pairs with Cytosine (C) through three hydrogen bonds, which are stronger and contribute to the overall stability of the DNA molecule.

Human Genome

  • 22 pairs of autosomal chromosomes and sex-determining chromosomes (XX or XY). The human genome comprises both autosomal and sex chromosomes, each carrying genes that determine various traits.

  • Mitochondrial DNA (hundreds of copies per cell) is also present; mothers pass it to their children. Mitochondrial DNA is located in the mitochondria, which are organelles responsible for energy production in cells. It is inherited solely from the mother.

  • Mitochondrial DNA is not as good for individualizing as nuclear DNA because it has a lower mutation rate and is inherited as a single unit (haplotype).

  • Cells in the body are diploid (two copies of each chromosome), except eggs and sperm, which are haploid (one copy). Diploid cells contain two sets of chromosomes, one from each parent, while haploid cells contain only one set.

  • Meiosis: Cell division where egg and sperm become haploid. Meiosis is a specialized cell division process that reduces the chromosome number by half to produce gametes (sperm and eggs).

  • Mitosis: Process where one diploid cell makes another diploid cell for growth or repair. Mitosis is a cell division process that produces two identical daughter cells, allowing for growth, repair, and maintenance of tissues.

  • Father's sperm (haploid) + mother's egg (haploid) = diploid cell. The fusion of sperm and egg during fertilization restores the diploid chromosome number in the offspring.

Genetics in Forensic DNA

  • The field of genetics is used to investigate genetic variation in the population to identify people. Forensic DNA analysis relies on understanding genetic variation in the population to differentiate individuals.

  • Population databases are needed because allele frequencies vary in different populations. Allele frequencies can differ significantly among different populations due to historical, geographic, and cultural factors.

Gregor Mendel's Work

  • Between 1856 and 1863, Gregor Mendel, a monk in Austria, used pea plants to track seven characteristics:

    • Seed shape, seed color, pod shape, pod color, flower color, flower location, and plant size. Mendel's experiments with pea plants laid the foundation for understanding the basic principles of inheritance.

  • Each characteristic had two options (e.g., round/wrinkled, yellow/green). These alternative forms of a trait are called alleles.

  • His work was rediscovered in 1900; he's considered the father of modern genetics. Mendel's laws of inheritance were initially overlooked but later recognized as fundamental principles in genetics.

  • Mendel found that each plant had two forms of each trait, one from each parent. This concept is known as the principle of segregation, where each individual carries two alleles for each trait, one inherited from each parent.

  • Alleles for different characteristics are independent of each other. This principle, known as the law of independent assortment, states that alleles of different genes assort independently of one another during gamete formation.

Law of Segregation

  • Allele pairs separate during gamete formation (sperm and eggs) and randomly unite at fertilization. During meiosis, the two alleles for each trait separate, so each gamete carries only one allele per trait.

  • Example: A parent with big A allele and small a allele (heterozygous) produces sperm cells with either big A or small a. Each sperm cell randomly receives either the big A or small a allele.

  • It's random which sperm reaches the egg first. The process of fertilization is random, with any sperm having an equal chance of fertilizing the egg.

  • The parent only gives one allele from each gene to the offspring at random. Each parent contributes only one allele for each trait to their offspring.

Law of Independent Assortment

  • During gamete formation, different pairs of alleles segregate independently of each other. The alleles of different genes assort independently during meiosis.

  • The allele a gamete receives for gene A has no impact on which allele it receives from gene B. This principle applies when genes are located on different chromosomes or far apart on the same chromosome.

  • Caveats:

    • Only true for genes on different chromosomes.

    • Or genes far enough apart on the same chromosome to undergo recombination during meiosis.

Gene Linkage and Recombination

  • Genes on the same chromosome close together are linked and do not undergo independent assortment. Linked genes tend to be inherited together because they are located close to each other on the same chromosome.

  • Genes far apart can undergo recombination (crossing over), acting as if they’re unlinked. Recombination can separate alleles of genes that are far apart on the same chromosome.

  • Crossing over or homologous recombination:

    • If genes are far enough apart, all four gamete combinations are equally possible (25% each). Genes that are far apart on the same chromosome behave as if they are unlinked due to frequent recombination events.

    • If genes are close, crossing over is rare, and gametes with parental combinations are more likely. Linked genes are more likely to be inherited together because crossing over is less frequent between them.

Human Genome and STR Loci

  • The human genome contains 20 FBI STR loci on 23 chromosomes. These STR loci are used in forensic DNA analysis for individual identification.

  • Some chromosomes have multiple markers (e.g., chromosome 2 has three). Having multiple markers on the same chromosome increases the power of discrimination in forensic DNA analysis.

  • These markers are far enough apart to be considered unlinked. The STR markers are strategically chosen to be unlinked to provide independent statistical information.

Inheritance Pattern

  • Dad and mom each have two alleles and pass one to the child. Each parent contributes one allele for each STR locus to their offspring.

  • This demonstrates independent assortment and law of segregation. The inheritance pattern follows Mendel's laws of segregation and independent assortment.

Hardy-Weinberg Equilibrium

  • Formula in population genetics independently discovered by Godfrey Hardy and Wilhelm Weinberg.

  • Allows estimating genotype frequencies from allele frequencies. The Hardy-Weinberg equilibrium principle is used to calculate the expected genotype frequencies in a population based on allele frequencies.

  • If there are two alleles, big A and small a:

    • Homozygous AA: p^2

    • Homozygous aa: q^2

    • Heterozygous Aa: 2pq

  • Allele frequency: the frequency of allele A or a in the population. Allele frequency refers to how common an allele is in a population.

  • Genotype frequency: The frequency of the genotype AA, Aa or aa in the population. Genotype frequency refers to how common a particular genotype is in a population.

  • Forensic DNA has more than two allele options (10-12), so it's useful to estimate genotype frequencies from allele frequencies. STR markers used in forensic DNA analysis have multiple alleles, making the Hardy-Weinberg equilibrium principle useful for estimating genotype frequencies.

  • A Punnett square explains the math behind p^2, 2pq, and q^2. A Punnett square is a diagram used to predict the possible genotypes of offspring in a genetic cross.

  • P+q=1

Relationship Between Allele and Genotype Frequencies

  • Ideal markers have a lot of heterozygosity in the population. Markers with high heterozygosity are more informative for individual identification.

  • Simplified example with big A and small a; in forensic DNA, there are 8-12 alleles per marker. STR markers used in forensic DNA analysis typically have multiple alleles.

Pedigree

  • Squares represent males; circles represent females. Pedigrees use standard symbols to represent individuals in a family.

  • Shows inheritance patterns across generations. Pedigrees are used to trace the inheritance of traits or genetic conditions through multiple generations.

  • Illustrates random allele combinations in children. Pedigrees can illustrate how alleles are combined randomly in offspring.

Forensic DNA: Criminal Justice System

  • Law enforcement collects evidence and submits it to the DNA lab. DNA evidence is collected at crime scenes and submitted to forensic labs for analysis.

  • The lab analyzes samples, and research determines new methods. Forensic labs analyze DNA samples using established protocols and also conduct research to develop new methods.

  • The output is a report to the prosecution and defense, potentially leading to a trial. The results of DNA analysis are presented in a report that is provided to both the prosecution and defense in a criminal case.

Principles of Forensic DNA Profiling

  • Each individual's genome is unique (except identical twins) and inherited from parents. DNA profiles are unique to each individual, except for identical twins, due to the inheritance of genetic material from both parents.

  • Markers are selected to differentiate individuals. STR markers are chosen for their ability to differentiate individuals in a population.

  • Hardy-Weinberg equilibrium is used to calculate statistical probabilities. Statistical analysis based on Hardy-Weinberg equilibrium is used to calculate the probability of a DNA match.

  • DNA typing is efficient and reproducible. DNA typing methods are designed to be efficient and produce reproducible results.

  • Markers are designed not to reveal information about race, disease, or phenotypic traits. STR markers used in forensic DNA analysis are generally chosen to avoid revealing sensitive information about individuals.

  • Newer tests may look at SNPs to predict hair color, eye color, and ancestry for investigative leads. SNPs (single nucleotide polymorphisms) can be used to predict physical traits and ancestry for investigative purposes.

Associations Using Biological Evidence

  • Goal: Compare question sample (crime scene) and known sample (suspect). Forensic DNA analysis involves comparing DNA profiles from crime scene samples and reference samples from suspects.

  • Isolate nuclear or mitochondrial DNA. DNA can be isolated from various biological samples, including blood, saliva, and hair.

  • Most labs focus on nuclear DNA. Nuclear DNA is the primary target for forensic DNA analysis due to its higher discriminatory power.

Sources of DNA

  • DNA profiles can be obtained from anything: Skin cells, nose, mouth, human remains, hair and tears on tissue paper. DNA can be extracted from a wide range of biological materials found at crime scenes.

Nuclear Genome

  • Consists of 3,000,000,000 base pairs (coding and non-coding DNA). The nuclear genome contains both coding (genes) and non-coding regions.

  • Forensic DNA focuses on non-coding regions with highly repetitive sequences (short tandem repeats). STR loci are located in non-coding regions of the genome and contain repetitive sequences.

  • STR loci are targeted for testing because people differ at these markers. The number of repeats at STR loci varies among individuals, making them useful for individual identification.

Short Tandem Repeat (STR) Marker

  • Primers bind to DNA for PCR amplification. Primers are short DNA sequences that bind to specific regions flanking the STR locus.

  • The repeat region (e.g., GATA repeated 11 times) is the target. The number of repeats at the STR locus is the basis for individual identification.

  • The number of repeats is reported (e.g., 11). The number of repeats is used to create a DNA profile for comparison.

  • Different people have different numbers of repeats. The variation in repeat numbers among individuals makes STR markers highly informative.

  • D7S820 locus, if it's a GATA pattern, there's three repeats here from the mother and four repeats from the father, so this would be a three allele and a four allele, and it would just be on the DNA profile, in terms of putting on the report, the D7 locus, the genotype is three, four, so it's simplified down from the repeat pattern down to just a three and four allele

DNA Profiling Steps

  • Crime committed, evidence collected, stored, and characterized. Proper collection, storage, and characterization of evidence are essential for accurate DNA analysis.

  • DNA extraction, quantification. DNA is extracted from the evidence and quantified to determine the amount of DNA present.

  • PCR amplification of STR markers. PCR (polymerase chain reaction) is used to amplify the STR markers for analysis.

  • Capillary electrophoresis (separation and detection). Capillary electrophoresis is used to separate and detect the amplified STR fragments.

  • Data interpretation and statistical calculations. The data is interpreted, and statistical calculations are performed to assess the significance of a DNA match.

  • Analysis, comparison, and evaluation (ACE principle). The ACE principle involves analysis, comparison, and evaluation of DNA profiles.

DNA Profile Comparison

  • Compare DNA profile from bloodstain to a reference profile from a suspect. DNA profiles from crime scene samples are compared to reference profiles from suspects.

  • Check if all alleles line up at all markers. A match is declared if all alleles at all STR markers align between the two profiles.

  • Inclusion/match or exclusion. The comparison results in either an inclusion (match) or exclusion.

  • Statistical weights are included in the report. Statistical weights are calculated to assess the significance of a DNA match.

Evaluation of Evidence

  • Exclusion: Question and known samples don't match. If the DNA profiles do not match, the suspect is excluded as the source of the evidence.

  • Inclusion/match: Alleles line up. If the DNA profiles match, the suspect is included as a potential source of the evidence.

  • Inconclusive: Poor DNA profile or complex mixture. Inconclusive results may occur due to poor DNA quality or complex mixtures of DNA from multiple individuals.

  • Paternity/kinship: The alleged father has at least one allele at all of the different locations, So he cannot be excluded as being the true father of the unknown child. Paternity testing involves comparing the DNA profiles of a child and an alleged father to determine if the alleged father can be excluded as the biological father.

Case Example: The Case of the Floating Feet

  • Shoes with human feet washed up on shore in the Lower Mainland/Vancouver Island since 2007.

  • The story gained international attention.

  • The BC coroner service investigated with a forensic anthropologist.

  • SFU researcher Gail Anderson investigated underwater carcasses.

  • Shoes and socks protected the feet, and Nike Air shoes had air pockets causing them to float once disarticulated.

  • All 15 feet in Canada have been identified using geographic information systems, forensic anthropology, and forensic DNA.