DNA Profiling (Ch#11) PDF

BSBT 2302 DNA ANALYSIS Fundamentals of Forensic Science Ch#11 Scientific evidence such as DNA typing does not convict anyone of a crime on its own. It is part of the network of evidence that, taken together, provides the proof that the judge or...

BSBT 2302 DNA ANALYSIS Fundamentals of Forensic Science Ch#11 Scientific evidence such as DNA typing does not convict anyone of a crime on its own. It is part of the network of evidence that, taken together, provides the proof that the judge or jury needs to conclude guilt or innocence. The Case: Colin Pitchfork The Case: Colin Pitchfork The Case: Colin Pitchfork The Case: Colin Pitchfork  Human Genome CHROMOSOMES, GENES & MARKERS A single copy of the human genome contains approximately three billion base pairs The Human Genome Project allowed a better understanding of genetic makeup and hence aided in forensic human identity testing The human genome consists of 22 matched pairs of autosomal chromosomes and 2 sex determining chromosomes 46 different chromosomes or 23 pairs of chromosomes Males as XY and Females as XX Most human identity-testing markers reside on autosomal chromosomes while sex- determining markers are on sex chromosomes Reference: Forensic DNA Typing: Biology & Technology Behind STR Markers By John Marshall Butler  Within the nucleus, the DNA is arranged into 46 structures (23 pairs) called chromosomes DNA is a nucleic acid polymer, composed of smaller monomeric units called nucleotides. Exists as two helices that wrap around each other like a spiral staircase Each nucleotide consists of three components: 1. Deoxyribose Sugar: A five-carbon sugar molecule. 2. Phosphate Group: Links the sugar molecules, forming the DNA backbone. 3. Nitrogenous Base: Adenine, Guanine, Cytosine & Thymine, Base pair specificity A=T, G≡C, The order of the base pairs contains a sort of blueprint, characteristic of a person DNA is located in two regions in a cell Both can be used for DNA typing 1. Nucleus 2. Mitochondria Unlike nuclear DNA, mitochondrial DNA is inherited only from the mother The Nature of DNA  VARIATIONS OF GENES: Alleles ▪ Each individual has two copies of each gene. One copy comes from the father, and the other from the mother ▪ Some traits are determined by a single gene at one locus others by multiple genes at several locations on chromosomes ▪ A particular locus (location) in a chromosome can have more than one form of DNA (polymorphism), then each form is called an allele. ▪ If a person inherits the same form of a gene from the mother and the father, that person is said to be homozygous for that gene ▪ For example, if a person inherits the type A form of the ABO gene from the mother and the father, the person is homozygous AA ▪ If a person receives different forms of the same gene (A and B), then that person is said to be heterozygous for that trait Historically, DNA fingerprinting was developed by Sir Alec Jeffrey at the University of Leicester in 1985 The first DNA typing method adopted by Forensic Biologists Not used any longer and is replaced by faster methods, using less biological material and providing higher resolution of discrimination such as PCR technique i.e, Polymerase Chain Reaction RFLP workflow: 1. DNA is extracted 2. Digested into small fragments called minisatellites or variable number tandem repeats (VNTRs) using Restriction Endonucleases 3. Separates them by gel electrophoresis depicting length polymorphism based on the number of repeating units in the VNTRs, used to discriminate a population of people 4. They are visualized by radio labeling or chemiluminescence. RFLP RESTRICTION FRAGMENT LENGTH POLYMORPHISM ▪ Polymorphic regions of DNA are identified ▪ Many hypervariable regions were identified across several loci called multilocus VNTRs ▪ Usually found between genes ▪ Restriction endonucleases cleave DNA in flanking regions adjacent to the VNTRs of interest. ▪ Restriction Endonucleases are designed to cut DNA at a specific sequence of bases. ▪ When DNA is cut with Restriction enzymes, the length of resulting DNA fragments varies between individuals due to differences in the number and location of restriction sites. These variations are detected and analyzed to create a DNA profile ▪ Usually, four to six of these highly polymorphic loci are analyzed, to generate individual profile RFLP RESTRICTION FRAGMENT LENGTH POLYMORPHISM  How RFLP Works? 1. Restriction Enzyme Digestion o The DNA is treated with restriction enzymes. o These enzymes recognize specific sequences and cut the DNA at or near these sites. o VNTRs remain intact within the resulting DNA fragments because the restriction sites are located outside the VNTR region. 2. Gel Electrophoresis o The digested DNA fragments are loaded onto an agarose gel and subjected to electrophoresis. o DNA fragments separate based on their size: smaller fragments move faster, and larger fragments move slower. o The VNTR-containing fragments are dispersed among other fragments of varying lengths.  How RFLP Works? 3. Southern Blotting (Transfer to Membrane) o The separated DNA fragments are denatured (converted into single strands) and transferred from the gel onto a nylon or nitrocellulose membrane through a Southern blotting process. o This ensures the fragments are immobilized and stable for further analysis. 4. Hybridization with VNTR-Specific Probes o A labeled DNA probe that is complementary to the VNTR region is introduced to the membrane. o This probe specifically binds (hybridizes) to the VNTR sequences because of sequence complementarity. o The rest of the DNA fragments on the membrane remain unbound by the probe.  How RFLP Works? 5. Detection of VNTR Fragments o The labeled probes are visualized using a detection system (e.g., autoradiography for radioactive probes or chemiluminescence for fluorescent probes). o Only the VNTR-containing fragments appear as bands on the final output, effectively separating them from the rest of the DNA.. 6. Comparison and Analysis o Compare the DNA profiles from the evidence and suspect(s) to determine a match or exclusion. LIMITATIONS 1. Difficulties in interpreting mixed samples 2. Problems with limited or degraded DNA  Single Locus VNTRs Homozygous: If an individual has the same number of repeats on both copies of a chromosome at a locus, the analysis produces one band of DNA fragments because both fragments are of the same size. Heterozygous: If the individual has a different number of repeats on each chromosome for a particular gene locus, the analysis produces two bands of DNA fragments, each corresponding to the size of the fragment from one chromosome. Find who is guilty among the suspects???  Restriction Enzymes Restriction Endonuclease Predominantly produced by bacteria Also known as Molecular Scissors Cleave DNA at specific sites called recognition sequences DNA sequences recognized by restriction enzymes are called palindromes Palindromes are base sequences that read the same on the two strands but in opposite directions. Named so because: Restrict infection of bacteria by certain viruses (i.e., bacteriophages), by degrading the viral DNA without affecting the bacterial DNA The purpose is to: 1. Defend against external threats (bacteriophage) 2. Ensure that the bacterium’s DNA is protected from being cut by its own enzymes.  Restriction Enzymes Restriction Endonuclease To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix. Catalyzing the hydrolysis (splitting of a chemical bond by addition of a water molecule) of the bond between adjacent nucleotides Can generate sticky ends or blunt ends  Nomenclature Restriction Endonucleases Each enzyme is named after the bacterium from which it was isolated, using a naming system based on bacterial genus, species, and strain. For Example: E – Escherichia: Genus co- coli: specific species R- RY13: strain I- First identified: order of identification in the bacterium RESTRICTION ENDONU CLEASES RESTRICTION ENZYMES  KEY DIFFERENCE BETWEEN VNTRs AND STRs In the number of nucleotides in a repeating sequence Minisatellites are a specific category of VNTRs, with repeat units ranging from 10–100 base pairs (bp) in length. These sequences are generally located in telomeric or sub-telomeric regions and were used in early DNA fingerprinting. Microsatellites are essentially the same as STRs, with shorter repeat units from 2–13 bp than minisatellites. Microsatellites/STRs are abundant in the genome, highly polymorphic, and widely used for forensic profiling, paternity testing, and population genetics.  KEY DIFFERENCE BETWEEN VNTRs AND STRs Because the repeats are right next to each other, without any intervening base pairs, these are referred to as tandem repeats NOMENCLATURE FOR DNA MARKERS NOMENCLATURE FOR DNA MARKERS EXAMPLE  VNTRs in Non-Coding Regions Majority of VNTR loci are located in the non-coding regions of DNA, such as: Introns: Non-coding regions within genes. Intergenic regions: DNA sequences between genes. VNTRs in these regions often have no direct impact on protein function but can affect: Gene regulation (e.g., transcription factor binding sites). Chromatin structure and epigenetics.  SHORT TANDEM REPEAT Today, most laboratories use short tandem repeats (STRs) typing method, which combines some of STR the attributes of both PCR and RFLP. STRs are typically located in non-coding regions of DNA STRs should exhibit a high degree of variability in the population i.e., are highly variable in repeat numbers among individuals Making it unlikely for individuals sharing the same STR profile by chance Should have a low mutation rate maintaining stability across generations Ideal markers for avoiding ethical concerns associated with coding DNA, which could potentially reveal personal traits. STR loci provide high discriminatory power in human identification, ensuring accuracy in forensic investigations. The number of STR loci analyzed varies depending on the country and database standards Generally, 13-20 STR loci are being typed for forensic purposes  SHORT TANDEM REPEAT ▪ Each multilocus genotype derived from the 13 loci being used is so rare that it is improbable that two people in the world STR would have the same exact type. ▪ Reliable population statistics have been developed for the various alleles of each of the 13 STRs ▪ Using STR typing, an identification is made between the DNA type of crime scene evidence and the possible suspect ▪ EXCEPT IN THE CASE OF IDENTICAL TWINS ▪ Among individuals, STR loci differ in the number of repeats they contain ▪ For a single loci, some individuals have shorter alleles while others might have longer alleles ▪ In addition to the repeat region, the PCR product might include a flanking region for primer annealing, adding 50- 100 bp  Population genetics ▪ A probability of a match between evidence and a potential suspect should be extremely low to be “significant” ▪ The FBI has historically used a threshold of 1 in 300 billion for a match to be considered a "match." This threshold is based on the estimated population of the United States. ▪ Analyzing more loci increases the discriminatory power of the DNA profile. The 13 core STR loci provide a high level of individualization. ▪ Population Data: Extensive research has been conducted to determine the frequencies of different alleles at each of the 13 core STR loci within various population groups (e.g., Caucasians, African Americans, Hispanics, Asians). ▪ These data are compiled into databases and used by forensic scientists. ▪ Population genetics  GENDER IDENTIFICATION 1. AMELOGENIN LOCUS ANALYSIS 2. Y-STR analysis  AMELOGENIN TYPING 1. AMELOGENIN LOCUS ANALYSIS Amelogenin is a locus on the sex- determination chromosome Males: One band from the X chromosome and another from the Y chromosome (6 base pairs longer). Females: Only one band due to two X chromosomes. Analyzed alongside STRs, appearing on the electropherogram.  AMELOGENIN TYPING Workflow 1. Primer Design: Primers targeting amelogenin on X and Y chromosomes are synthesized with incorporated fluorescent dyes. 2. PCR Amplification: Dye-labeled primers anneal to target DNA, and DNA polymerase extends them, incorporating the dyes into the amplicons. 3. Capillary Electrophoresis: Dye-labeled fragments are separated by size. Laser excitation at the detection window causes the dyes to emit light. 4. Signal Detection: Emitted light is detected and converted into an electrical signal, generating peaks on the electropherogram.  Y-STR analysis STRs on the Y chromosome (found only in males). Useful for small, degraded, or mixed samples with female DNA dominance. Produces a haplotype, making it less informative than regular STR analysis. Y-chromosome undergoes less recombination during inheritance, establish paternal relationships Thus share same paternal haplotype ie., same set of STR markers inherited as a block. Less discriminating than standard STR analysis  “HAPLOTYPE” A haplotype (short for "haploid genotype") refers to a set of specific genetic variations or markers that are inherited together on a single chromosome from one parent. Haplotypes are particularly useful in genetics and genomics for identifying patterns of inheritance, tracing ancestry, and studying population genetics. Haplotype vs. Genotype Genotype: Refers to the specific genetic makeup at a single locus (or a set of loci) on both chromosomes. Haplotype: Refers to the specific genetic makeup on a single chromosome (inherited from one parent).  Y-STR MARKERS Relative positions of the commonly used Y-STR markers on the Y chromosome  The PCR Process PCR Reaction Components: 1. Reaction Buffer: Provides optimal pH and salt conditions for the polymerase. 2. dNTPs: Deoxynucleoside triphosphates (dATP, dTTP, dCTP, dGTP) - the building blocks of DNA. 3. Taq Polymerase: Heat-stable DNA polymerase enzyme (commonly used). 4. DNA template. 5. Locus specific primers  The PCR Process PCR Thermal Cycling: 1. Thermal Cycler: Instrument that precisely controls temperature changes. 2. Three Steps: Denaturation (~95°C), Annealing (~55-60°C), and Extension (~72°C). 3. Cycles: Multiple cycles of denaturation, annealing, and extension are performed to amplify DNA exponentially (25-40 cycles). 4. Final Extension: ~72°C for ~ 10 min Alleles scored at 15 STR loci in the child and the mother and the alleged father. At each locus, the child received one STR allele from his mother and the other from his father (alleged father).  Nuclear DNA Deoxyribonucleic acid (DNA) is a molecule that is found in nearly all cells. DNA is located in two regions in a cell: the nucleus and mitochondria Notable exceptions are red blood cells, which have neither nucleus nor mitochondria  Variable Loci Example: ABO markers Polymorphic loci exhibit variation among members of a population. These variable loci are purposely chosen The more variation there is at a locus, the more discriminating power it holds for the identification analysis. For example, in the Caucasian population, the ABO blood system is present as: a. Type A blood is 42% b. Type O is about 43% c. Type B is about 10% d. Type AB in about 5% Thus the locus for ABO blood type does show some variation but, by itself, isn’t very discriminating, since even its rarest form would still include 5% of individuals as being the source of a blood sample.  Variable Loci EXAMPLE: First Typing Marker DQA1 The first marker to be amplified and used forensically was DQα (now called DQA1). Encodes a protein in Human Leukocyte Antigen (HLA) complex HLA complex of genes located on chromosome 6 Known to present antigen for T-cell activation or antibody production The variability of this complex allows the identification of wide range of foreign antigens  Mitochondrial DNA mtDNA Like nuclear DNA, mtDNA is present in all cells and are known as the energy mediator of the cell Structure: Circular DNA with 16,569 base pairs coding for 37 genes. Quantity: up to 10 copies of mtDNA/mitochondrion; Hundreds or thousands of mtDNA copies vs. 2 copies of nuclear DNA per cell. Their number per cell can vary significantly depending on the cell type and its energy requirements Non-coding Region: Contains a 1,100 base-pair non-coding region with two hypervariable regions HVR1 and HVR2 that has higher mutation rate over generations Use in Degraded samples: Large mtDNA copy numbers make it ideal for typing degraded, old, or low- DNA samples e.g., Old bones, tooth, and hair with no root. Maternal Inheritance: Inherited exclusively from the mother. Shared by all maternal descendants, either identical or very similar. Application: High variation between unrelated individuals aids in forensic typing. A powerful tool for tracing family lines back through the maternal side Drawback: Since only two hypervariable regions in mtDNA, the population statistics are not nearly as discriminating as with nuclear DNA. Mitochondrial DNA mtDNA  Mitochondrial DNA mtDNA mtDNA analysis generally requires DNA sequencing Allowing determination of the entire base pair sequence in the two hypervariable regions, rather than relying on length polymorphism Detects sequence polymorphism rather than length polymorphism Less discriminating among individuals sharing similar maternal ancestries Used in resolving disputes may occur in cases of surrogacy, adoption, or rare medical errors (e.g., mix-ups at birth). Single Nucleotide Polymorphisms (SNPs) ▪ A type of sequence polymorphism ▪ SNPs are variations in a single DNA nucleotide that occur at specific positions in the genome. ▪ While identical twins share most of their SNPs, subtle differences can arise during early development due to random mutations. ▪ By analyzing a large number of SNPs, researchers can identify unique patterns in each twin, allowing for their differentiation.  Epigenetics Identical twins, despite having the same DNA, can develop different epigenetic patterns over time and thus can be differentiated on their basis Changes in gene expression but not due to alterations in underlying DNA sequence Changes influenced by environmental factors and life experiences Techniques like DNA methylation analysis used for identification of epigenetic differences Techniques involved: DNA Methylation Microarrays & Bisulfite Sequencing (Gold Standard)  DNA Methylation Typically, DNA methylation silences gene expression. A methyl group (CH3) is added by DNA methyltransferase (DNMT) enzyme, specifically to the 5th carbon of cytosine followed by guanine (CpG sites). Methylated DNA attracts proteins that block access to the gene for transcription factors, preventing gene activation.  Whole Genome Sequencing ▪ Complete the DNA sequence of an individual. ▪ Identifies even the smallest genetic variations between individuals, including rare mutations unique to either twin ▪ Currently expensive and time-consuming, making it less practical for routine forensic analysis  How can Identical Twins be Differentiated by DNA profiling??? Identical twins, also known as monozygotic twins, are formed from a single fertilized egg that splits into two. Advancements in DNA analysis techniques have made it possible to differentiate between them some methods that can be used are: 1. Single Nucleotide Polymorphisms (SNPs) 2. Epigenetics 3. Whole Genome Sequencing (gold standard, detects rare variants SNPs or copy number variations) 4. Mitochondrial DNA (mtDNA) Analysis (Less efficient)  Twin Crimes: Case Study In 2010, Georgia-based twin sisters Jasmiyah and Tasmiyah Whitehead, then 16, tried to fool investigators into thinking they’d stumbled on the crime scene of their dead mother. In actuality, they were the killers. (Rockdale County Sheriff's Office)  Twin Crimes: Case Study In a 2012 case in southern France, six women were raped by a single man. DNA evidence implicated identical twin brothers, Yoan and Elvin Gomis. Victims couldn't identify their attacker, and DNA tests were inconclusive due to the twins' identical genetic profiles. Both men were detained for 10 months before Yoan Gomis confessed to the crimes.  FAMILIAL DNA SEARCHES 1. DNA Analysis: Crime scene DNA is compared against profiles in a database. 2. Partial Match Identification: Instead of looking for an exact match, the system identifies profiles that share a significant portion of genetic markers, indicating a likely biological relationship. 3. Investigative Follow-Up: Once a partial match is found, investigators may identify the individual’s family members whose DNA is in the database and determine if any of them could be the potential suspect. COMPARISON OF DNA TYPING TECHNOLOGIES  Combined DNA Index System (CODIS) Database FBI had created a three-tiered: local, state, and national database It contains nearly six million DNA profiles CODIS consists of three sets of databases 1. National CODIS system (NDIS) by the FBI 2. State CODIS systems (SDIS) 3. Local databases (LDIS) by many large cities Data Flow: DNA profiles are first collected at the local level, then fed into state databases, and finally into the national database, allowing searches at different levels. Access Restrictions: Only ISO or ASCLD-accredited crime laboratories have access to CODIS, limiting data use by academic researchers.  Types of CODIS Database 1. Forensic Database: Contains DNA profiles from crime scenes, with unknown sources. 2. Offender Database: Includes DNA profiles of criminal offenders and sometimes arrestees for felonies and misdemeanors. 3. Missing Persons Database: Aims to be comprehensive and nationwide to help identify missing individuals, especially those who may have crossed state lines. Pre-Database Collection of DNA Ethical & Privacy Concerns Collecting DNA from individuals who are not suspects can raise privacy issues and ethical questions about consent and data usage. Resource Intensive: Collecting, processing, and analyzing DNA from a large group can be expensive and time-consuming This approach is typically seen as a last resort when other investigative methods fail  How does Forensic DNA Profiling ensure accuracy in investigations? 1. Chain of Custody: Proper collection, labeling, and handling of samples ensure integrity. 2. Quality Controls: Laboratories follow strict protocols like ISO/IEC 17025 accreditation. 3. Statistical Validation: Likelihood ratios and probability metrics help quantify the reliability of a match.  How does Forensic DNA Profiling ensure accuracy in investigations? 4. Blinding: Analysts are often blinded 5. Replicability: Independent to case details to reduce bias. verification of results by different analysts or labs.  CONCLUDING REMARKS The Pitchfork case, being the first of its type in criminal investigation, did not rely on any population statistics for interpretation. It was a decade or more before such data were organized and collected. No claim was made by Dr. Jeffries or the other scientists that Pitchfork was the only person in the world, who possessed the DNA characteristics, that were revealed by the tests done in this case. The other circumstances of the case—that Pitchfork was local and that the person he paid to impersonate him bragged about it to friends in a bar— were all that was necessary to implicate Pitchfork.