DNA Polymorphisms and Human Identification
TYPES OF POLYMORPHISMS
Polymorphisms are variations in DNA sequences shared by a certain percentage of a population.
These sequences can range from a single base pair to thousands of base pairs.
The probability of polymorphic DNA in humans is high due to the large size of the human genome, where 98% does not code for genes.
Types of Polymorphisms
SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs)
Occur in gene-coding regions or intergenic sequences.
More frequent in some genomic areas, like the human leukocyte antigen (HLA) locus.
HLA locus: highly polymorphic region coding for peptides that establish the immune system's self-identity.
Long Interspersed Nucleotide Elements (LINEs)
Highly repeated sequences (6 to 8 kbp).
Contain RNA polymerase promoters and open reading frames related to retrovirus reverse transcriptase.
Short Interspersed Nucleotide Elements (SINEs)
SINEs are 0.3 kbp in size and present in over 1,000,000 copies per genome.
Includes Alu elements, named for AluI restriction enzyme recognition sites.
Over 1 million Alu elements account for almost 11% of the human genome.
Most transcribed genes contain Alu elements in their introns.
Alu elements have cryptic splice and polyadenylation sites that can become activated through mutations, leading to alternative splicing or premature translation termination.
LINEs and SINEs are mobile or transposable elements, copied and spread by recombination and reverse transcription, potentially forming pseudogenes.
. SHORT TANDEM REPEATS (STRs) and VARIABLE-NUMBER TANDEM REPEATS (VNTRs)
Shorter blocks of repeated sequences undergo expansion or shrinkage through generations.
SNPs, larger sequence variants, and tandem repeats can be detected by observing changes in the restriction map of a DNA region.
Analysis of restriction fragments by Southern blot reveals restriction fragment length polymorphisms (RFLPs).
RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPs)
Analysis of restriction fragments by Southern blot
One or more nucleotide changes that affect the size of restriction enzyme products
Historical Highlights
1920s: Scientists realized that blood type (A, B, AB, or O) is inherited and could be used for parentage testing.
This limited testing could only exclude a falsely alleged father.
Later, other proteins on the surface of the red blood cell (Rh, Kell, and Duffy blood group systems) were introduced.
The power of these serological tests was only marginally better than that of the ABO system.
Forty years later, the polymorphic HLAs were implemented for parentage and identity testing, coupled with the ABO and serological testing.
RFLP TYPING
Original DNA targets used for gene mapping, human identification, and parentage testing.
First polymorphic RFLP was described in 1980.
Observed as differences in the sizes and number of fragments generated by restriction enzyme digestion of DNA.
Nucleotide changes may also destroy, change, or create restriction enzyme sites, altering the number of fragments.
The first step in using RFLPs is to construct a restriction enzyme map of the DNA region under investigation.
Once the restriction map is known, the number and sizes of the restriction fragments of a test DNA region cut with restriction enzymes are compared with the number and sizes of fragments expected based on the restriction map
Polymorphisms are detected by observing fragment numbers and sizes different from those expected from the reference restriction map.
RFLP typing in humans required the use of the Southern blot technique.
DNA was cut with restriction enzymes, resolved by gel electrophoresis, and blotted to a membrane.
RFLP TYPING (continued)
One consequence of genetic diversity is that a single locus (gene or DNA region) will have several versions, or alleles.
Human beings are diploid with two copies of every locus.
Each person has two alleles of each locus.
If alleles are the same, the locus is homozygous.
If the two alleles are different, the locus is heterozygous.
Same allele = HOMOZYGOUS
Different alleles = HETEROZYGOUS
The uniqueness of the collection of polymorphisms in each individual is the basis for human identification at the DNA level.
Detection of RFLP by Southern blot made positive paternity testing and human identification possible for the first time.
RFLP protocols for human identification in most North American laboratories used the restriction enzyme HaeIII for fragmentation of genomic DNA
Many European laboratories used the HinfI enzyme.
All of these enzymes cut DNA frequently enough to reveal polymorphisms in multiple locations throughout the genome.
Genetic Mapping With RFLPs
Polymorphisms are inherited in a Mendelian fashion, and the locations of many polymorphisms in the genome are known.
Therefore, polymorphisms can be used as landmarks, or markers, in the genome to determine the location of other genes
Formal statistical methods are used to determine the probability that an unknown gene is located close to a known marker in the genome.
This is the basis for linkage mapping and one of the ways genetic components of disease are identified.
Historical Highlights: Mary Claire King
Mary Claire King used RFLP to map one of the genes mutated in inherited breast cancer.
Following extended families with high incidence of breast and ovarian cancer, she found particular RFLP always present in affected family members.
Because the location in the genome of the RFLP was known (17q21), the BRCA1 gene was thereby mapped to this position on the long arm of chromosome 1.
RFLP and Parentage Testing
In diploid organisms, chromosomal content is inherited, half from each parent. This includes the DNA polymorphisms located throughout the genome.
The fragment sizes of an individual are a combination of those from each parent.
In a paternity test, the alleles or fragment sizes of the offspring and the mother are analyzed.
The remaining fragments (the ones that do not match the mother) have to come from the father
Of the two alleged fathers shown, only one could supply the fragments not supplied by the mother. In this example, only two loci are shown. A parentage test requires analysis of at least eight loci. The more loci tested, the higher the probability of positive identification of the father.
Human Identification Using RFLPs
The first genetic tool used for human identification was the ABO blood group antigens. Although this type of analysis could be performed in a few minutes, the discrimination power was low.
With only four possible groups, this method was only good for exclusion (elimination) of a person and was informative only in 15% to 20% of cases.
The initial use of DNA as an identification tool relied on RFLP detectable by Southern blot.
The insertion or deletion of nucleotides occurs frequently in repeated sequences in DNA. Tandem repeats of sequences of all sizes are present in genomic DNA
DNA Fingerprinting With RFLP
The first human DNA profiling system was introduced by the United Kingdom Forensic Science Service in 1985 using Sir Alec Jeffrey’s Southern blot multiple-locus probe (MLP) -RFLP system
This method utilized three to five probes to analyze three to five loci on the same blot.
Results of probing multiple loci at once produced patterns that were highly variable between individuals but that required some expertise to optimize and interpret.
In 1990, single-locus probe (SLP) systems were established in Europe and North America
Analysis of one locus at a time yielded simpler patterns, which were much easier to interpret, especially in cases where specimens might contain a mixture of DNA from more than one individual
P-based probe system could take 5 to 7 days to yield clear results
RFLP is an example of a continuous allele system in which the sizes of the fragments define alleles. Therefore, precise band sizing was critical to the accuracy of the results
This process established the likelihood of the same genotype occurring by chance. The probability of two people having the same set of RFLP, or profile, becomes lower and lower as more loci are analyzed.
Genetic Mapping With RFLPs (Historical Highlights: Professor Sir Alec John Jeffreys)
Professor Sir Alec John Jeffreys, a British geneticist, first developed techniques for genetic profiling, or DNA fingerprinting, using RFLP to identify humans.
The technique has been used in forensics and law enforcement to resolve paternity and immigration disputes.
The method can also be applied to nonhuman species, for example, in wildlife population genetic
The first application of this DNA technique was in a regional screen of human DNA to identify the rapist and killer of two girls in Leicestershire, England, in 1983 and 1986. Colin Pitchfork was identified and convicted of murder after samples taken from him matched semen samples taken from the two victims.
STR TYPING BY PCR
The first commercial and validated typing test based on polymerase chain reaction (PCR) specifically for forensic use was the HLA DQ alpha system, now called DQA1, developed in 1986
This system could distinguish 28 DQA1 types
The PM system is a set of primers complementary to sequences flanking STRs, or microsatellites. STRs are similar to VNTRs (minisatellites) but have repeat units of 1 to 7 bp.
STRs contain repeat units with altered sequences, or microvariants, repeat units missing one or more bases of the repeat. These differences have arisen through mutation or recombination events.
In contrast to VNTRs, the smaller STRs are efficiently amplified by PCR, easing specimen demands significantly. Long, intact DNA fragments are not required to detect the STR products; therefore, degraded or otherwise less-than-optimal specimens are potentially informative
Although STRs with 4- and 5-bp repeat units are highly informative and efficiently amplified, they are subject to naturally occurring genetic events. Loss or gain of repeats or parts of repeat units, as well as mutations within repeat units, are very rare occurrences. Because at least 8 to more than 20 loci are included in STR applications
STR alleles are identified by PCR product size. Primers are designed to produce amplicons of 100 to 400 bp in which the STRs are embedded
The sizes of the PCR products are influenced by the number of embedded repeats. If one of each primer pair is labeled with a fluorescent marker, the PCR product can be analyzed in fluorescent detection systems. Silver-stained gels may also be used; however, capillary electrophoresis with fluorescent dyes is the preferable method, especially for high- throughput requirements
A further development of STR analysis was the design of mini-STR
Compared with standard STR products, the small amplicons are more efficiently produced from such challenging starting material as fixed tissue 11 and degraded specimens
Y-STR was developed for surname testing and forensic identification of male offenders or victims. This primer set only amplifies STR located on the Y chromosome
STR Analysis
To identify STR alleles, test DNA is mixed with the primer pairs, buffer, and polymerase to amplify the test loci.
Following amplification, each sample PCR product is combined with allelic ladders (sets of fragments representing all possible alleles of a repeat locus) and internal size standards (molecular-weight markers) in formamide for electrophoresis.
RFLPs and VNTRs, STRs are discrete allele systems in which a finite number of alleles is defined by the number of repeat units in the tandem repeat
The allelic ladders in these reagent kits allow accurate identification of the sample alleles
capillary electrophoresis is faster and more automated than gel electrophoresis, a single run through a capillary of single dye-labeled products can resolve only loci whose allele ranges do not overlap.
As in RFLP testing, an STR “match” is made by comparing profiles (alleles at all loci tested) followed by probability calculations.
STR Nomenclature
International Society for Forensic Genetics recommended nomenclature for STR loci in 1997. 15 STRs within genes are designated according to the gene name.
STR TH01 is located in intron 1 of the human tyrosine hydroxylase gene on chromosome 11, and TPOX is located in intron 10 of the human thyroid peroxidase gene on chromosome 2.
Non-gene-associated STRs are designated by the D#S# system.
In this system, the “D” stands for DNA; the following number designates the chromosome where the STR is located (1-22, X or Y).
“S” refers to a unique segment, followed by a number registered in the International Genome Database (GDB).
Gender Identification
Amelogenin locus is a very useful marker often analyzed along with STR
The amelogenin gene, which is not an STR, is located on the X and Y chromosomes. The function of its encoded protein is required for embryonic development and tooth maturation
Amplification and electrophoretic resolution reveal two bands or peaks for males (XY) and one band or peak for females
Genotyping
DNA testing results in peak or band patterns that must be converted to genotype
Microvariant alleles containing partial repeat units are indicated by the number of complete repeats followed by a decimal point and then the number of bases in the partial repeat
The genotype, or profile, of a specimen is the collection of alleles in all the loci tested.
Genetic concordance is a term used to express the situation where all locus genotypes (alleles) from two sources are the same.
Technical artifacts such as air bubbles, crystals, and dye blobs, as well as sample contaminants, temperature variations, and voltage spikes, can interfere with consistent band migration during electrophoresis
Stutter is another anomaly of PCR amplification, in which the polymerase may miss a repeat during the replication process, resulting in two or more different species in the amplified product
bin can be thought of as an uncertainty window surrounding the mean position (size) of multiple runs of each peak or band.
Binning is the collection of all peaks or bands within a characteristic distribution of positions and areas
The fixed-bin approach is an approximation of the more conservative floating-bin approach. 18 An alternative assessment of allele certainty is the use of locus- specific brackets
Matching of Profiles
The more loci analyzed, the higher the probability that the locus genotype positively identifies an individual (match probability)
These criteria are based on validation studies and results reported from other laboratories
Periodic external proficiency testing is performed to confirm the accuracy of test performance.
Hardy–Weinberg equilibrium, or the Hardy–Weinberg law. The population frequency of two alleles, p and q, can be expressed mathematically as
The frequency of a set of alleles or a genotype in a population is the product of the frequency of each allele separately (the product rule ). The product rule can be applied because of linkage equilibrium
Allele frequencies differ between subpopulations or ethnic groups
The likelihood ratio is the comparison of the probability that the two genotypes came from the same person with the probability that the two genotypes came from different persons, taking into account allele frequencies and linkage equilibrium in the population
Sir Alec Jeffreys’ DNA profiling was the basis for the National DNA Database (NDNAD) launched in Britain in 1995
The DNA profile of anyone convicted of a serious crime is stored on a database
The National DNA Index System (NDIS) is the federal level of the CODIS used in the United States. There are three levels of CODIS: the Local DNA Index System (LDIS), State DNA Index System (SDIS), and NDIS.
Allelic Frequencies in Paternity Testing
Paternity test is designed to choose between two hypotheses:
a. The test subject is not the father of the tested child (),
b. or the test subject is the father of the tested child ()
Peter Gill developed a forensic DNA identification method for minimal samples called lowcopy- number analysis
Low-copy-number analysis is reportedly performed on less than 100 pg DNA (about 16 diploid cells) Paternity index, or likelihood of paternity, is calculated for each locus in which the alleged father and the child share an allele Paternity index is an expression of how many times more likely the child ’ s allele is inherited from the alleged father than by another man in the general population
Allelic Frequencies in Paternity Testing (continued)
Combined paternity index (CPI), which summarizes and evaluates the genotype information
Probability of paternity, a number calculated from the combined paternity index (genetic evidence) and prior odds (nongenetic evidence)
Sibling Tests
A sibling test is a more complicated statistical analysis than a paternity test.
kinship index, sibling index, or combined sibling index - ratio generated by sibling test.
A full sibling test is a determination of the likelihood that two people tested share a common mother and father.
A half-sibling test is a determination of the likelihood that two people tested share one common parent (mother or father).
avuncular testing, which measures the probabilities that two alleged relatives are related as either an aunt or an uncle of a niece or nephew.
Y-STR
Y-STRs are represented only once per genome and only in males.
A set of Y-STR alleles comprises a haplotype, or series of linked alleles always inherited together.
exploited for forensic, lineage, and population studies as well as kinship testing.
The Y-STR/paternal lineage test can determine whether two or more males have a common paternal ancestor
Y-STR (continued)
Y-STRs have been utilized in forensic tests where the evidence consists of a mixture of male and female DNA, such as semen, saliva, other body secretions, or fi ngernail scrapings.
Lineage testing over several generations is made possible by this low mutation rate of Y chromosome.
Y-STRs have microvariant alleles containing incomplete repeats and alleles containing repeat sequence differences.
Matching with Y-STRs
Haplotype diversity (HD) is calculated from the frequency of occurrence of a given haplotype in a tested population.
Discriminatory capacity (DC) is determined by the number of different haplotypes seen in the tested population and the total number of samples in the population
The European Y chromosome typing community has established a set of Y-STR loci termed the minimal haplotype
An “extended haplotype” includes all of the loci from the minimal haplotype plus the highly polymorphic dinucleotide repeat YCAII
Matching with Y-STRs (continued)
The results of a Y typing might be reported accompanied by the number of observations or frequency of the analyzed haplotype in a database of adequate size.
Y-chromosome haplotypes can be used to exclude paternity.
Paternity index is calculated in a manner similar to the autosomal STR analysis.
6 Y-STR alleles are tested and match between the alleged father and child. If the haplotype has not been observed before in the population, 0/1,200, and the haplotype frequency will be 1/1,200, or 0.0008333.
The PI is then 1/0.0008333 = 1,200. With a prior probability of 0.5, the probability of paternity is or 99.9%
Y-STRs as marker loci for Y-chromosome, or surname, tests are used to determine ancestry.
LINKAGE ANALYSIS
Three basic approaches are used to map genes: family histories, population studies, and sibling analyses.
Family history and analysis of generations of a single family for the presence of a particular STR allele in affected individuals is one way to show association.
linkage analysis is to look for gene associations in large numbers of unrelated individuals in population studies.
Sibling studies are another type of linkage analysis. Monozygotic (identical) and dizygotic (fraternal) twins serve as controls for genetic and environmental studies.
BONE MARROW ENGRAFMENT TESTING USING DNA POLYMORPHISMS
Bone marrow transplantation is a method used to treat malignant and nonmalignant blood disorders as well as some solid tumors.
autologous (from self)
allogeneic transplants (between two individuals)
Donor cells are supplied as bone marrow, peripheral blood stem cells (hematopoietic stem cells), or umbilical cord stem cells.
HLA typing - to assure successful establishment of the transplanted donor cells, the immune compatibility of the donor and recipient is tested prior to the transplant.
BONE MARROW ENGRAFMENT TESTING USING DNA POLYMORPHISMS (continued)
In allogeneic transplant strategies, high doses of therapy completely remove the recipient bone marrow, particularly the stem cells that give rise to all the other cells in the marrow (conditioning).
The allogeneic stem cells are then expected to reestablish a new bone marrow in the recipient (engraftment).
Sub-myeloablative transplant procedures or mini-transplants used to reduce the toxicity of this procedure.
This process also imparts a graft-versus leukemia (GVL) or graft-versus-tumor (GVT) effect, which is a process closely related to graft-versus host disease (GVHD).
BONE MARROW ENGRAFMENT TESTING USING DNA POLYMORPHISMS
Allogeneic Transplantation
The first phase is donor matching (HLA).
Donors may be known or related to the patient or anonymous unrelated contributors (matched unrelated donor (MUD).
The National Marrow Donor Program (NMDP) maintains a database of people who have voluntarily submitted their HLA types and are willing to serve as potential donors.
After conditioning and infusion with the donor cells, the patient enters the engraftment phase.
The engraftment of donor cells in the recipient must be monitored (90 days).
Chimera - an individual carrying two population of cells that arose from the same zygote.
DNA typing has become the method of choice for engraftment monitoring.
BONE MARROW ENGRAFMENT TESTING USING DNA POLYMORPHISMS (continued)
In the laboratory, there are two parts to engraftment/ chimerism DNA testing.
Before the transplant, several polymorphic loci in the donor and recipient cells are screened to find at least one informative locus.
Noninformative loci are those in which the donor and the recipient have the same alleles.
The second part of the testing process is the engraftment analysis.
BONE MARROW ENGRAFMENT TESTING USING DNA POLYMORPHISMS (continued)
Pretransplant analysis and engraftment were measured in early molecular studies by amplification of small VNTRs and resolution of amplified fragments on polyacrylamide gels with silver-stain detection.
PCR amplification is the preferred method of STRs, resolution by capillary electrophoresis, and fluorescent detection.
Alternative method: qPCR of SNP.
advantage of higher throughput and lower sample requirements.
PSTR Testing
Donor and recipient DNA for allele screening prior to transplant can be isolated from blood or buccal cells.
The lower limit of 1 ng of DNA is reportedly sufficient for the screening of multiple loci; however, 10 ng is a more practical lower limit.
Multiple loci can be screened simultaneously using multiplex PCR.
Primer sets that specifically amplify Y-STR may also be useful for sex-mismatched donor–recipient pairs.
Split chimerisms - isolated granulocytes may show full chimerism, whereas the T-cell fraction still shows mixed chimerism.
PSTR Testing (continued)
Although the capillary electrophoresis used for this method is the same as that used for sequence analysis, measuring peak sizes and peak areas is distinguished from sequence analysis as fragment analysis.
Automatic detection will generate an electropherogram.
Informative and noninformative loci will appear as nonmatching or matching donor and recipient peaks.
Optimal loci for analysis should be clean peaks without stutter, especially stutter peaks that co-migratewith informative peaks, nonspecifi c amplifi ed peaks (mis- primes), or other technical artifacts.
PSTR Testing (continued)
Ideally, the chosen locus should have at least one recipient informative allele.
The amelogenin locus supplies a recipient-informative locus if the recipient is male and the donor is female.
Good separation of the recipient and donor alleles is desirable for ease of discrimination in the post-transplant testing
After the transplant, the recipient is tested on a schedule determined by the clinician or according to consensus recommendations.
With nonmyeloablative or reduced-intensity pretransplant protocols, an example schedule would be testing at 1, 3, 6, and 12 months.
Bone marrow specimens can most conveniently be taken at the time of bone marrow biopsy following the transplant, with blood specimens taken in intervening periods.
3 to 5 mL of bone marrow or 5 mL of blood is more than sufficient for analysis
5 to 7 mL bone marrow, 10 to 20 mL blood may be required.
Post-Transplant Engraftment Testing
Quantification of the percentage of recipient and donor cells post-transplant is performed using the informative locus or loci selected during the pretransplant informative analysis.
Peaks are generated by the emission from the fl uorescent dyes attached to the primers and thus to the ends of the PCR products collected as each product migrates past the detector.
The fluorescent signal is converted into fluorescence units by the computer software.
The software displays the PCR products as peaks of fluorescence units ( y -axis) versus migration speed ( x - axis).
Post-Transplant Engraftment Testing (continued)
homozygous or heterozygous donor and recipient peaks with no shared alleles, the percentage of recipient cells is equal to R/(R + D)
R is the height or area under the recipient-specifi c peak(s)
D is the height or area under the donor-specific peak(s).
Chimerism/engraftment results are reported as the percentage of recipient cells and/or percentage of donor cells in the bone marrow, blood, or cell fraction.
The first determination to be made from engraftment testing is whether donor engraftment has occurred and, secondly, whether there is split chimerism.
QUALITY ASSURANCE FOR SURGICAL SECTIONS USING STR
The molecular diagnostics laboratory can assist in ensuring that surgical tissue sections are properly identified and not contaminated.
During processing of tissue specimens, microscopic fragments of tissue may persist in paraffin baths (floaters).
STR identification can confirm the origin of tissue.
SINGLE NUCLEOTIDE POLYMORPHISMS
The traditional defi nition of polymorphism requires that the genetic variation be present at a frequency of at least 1% of the population.
mapping studies achieved denser coverage of the genome. 45 The most definitive way to detect SNPs has been by direct sequencing.
Next generation sequencing has greatly accelerated both the discovery and detection of SNPs.
A familiar example is the SNP responsible for the formation of hemoglobin S in sickle cell anemia.
classified according to location, relation to coding sequences, and whether they cause a conservative or nonconservative sequence alteration
SNP databases such as dbSNP, dbVar, ClinVar, and others are collections of DNA sequence variants used as a reference for screening genomic sequencing data.
The Human Haplotype Mapping (HapMap) Project
Its goal was to develop a haplotype map of the human genome
It is used to identify common disease associations and patterns of human DNA sequence variation.
MITOCHONDRIAL POLYMORPHISM
The two strands of the circular mitochondrial DNA (mtDNA) chromosome have an asymmetric distribution of Gs and Cs generating a G-rich heavy (H) and a C-rich light (L) strand.
Each strand is transcribed from a control region starting at one predominant promoter, P L on the L strand and P H on the H strand, located in sequences of the mitochondrial circle called the displacement (D)-loop.
The D-loop forms a triple-stranded region with a short piece of H-strand DNA, the 7S DNA, synthesized from the H strand. Bidirectional transcription starts from P L on the L-strand and P H1 and P H2 on the H-strand.
Mature mitochondrial RNAs, 1 to 17, are generated by cleavage of the polycistronic (multiple- gene) transcript at the location of the tRNA genes.
MITOCHONDRIAL POLYMORPHISM
Mitochondrial genome has two noncoding regions that vary in DNA sequence, the hypervariable region 1 and the hypervariable region 2, or HV1 and HV2
The reference mtDNA hypervariable region is the sequence published initially by Anderson, called the Cambridge reference sequence, the Oxford sequence, or the Anderson reference.
Mitochondrial nucleotide sequence data are divided into two components:
Forensic
consists of anonymous population profiles
used to assess the extent of certainty of mtDNA identifi cations in forensic casework.
Public
provide information on worldwide population groups not contained within the forensic data and can be used for investigative purposes.
OTHER IDENTIFICATION METHODS
Protein-Based Identification
Protein polymorphisms are in the form of amino acid sequence variations.
It has been proposed that protein polymorphisms may serve as supportive confirmation or even an alternative for DNA identification results.
Nonsynonymous DNA polymorphisms produce single-amino-acid polymorphisms in proteins.
Peptide variants in these proteins can be identified using liquid chromatography followed by mass spectrometry.
Proteins isolated from test samples are reduced, alkylated, and digested with trypsin, and the resulting peptides are resolved by liquid chromatography.
Software matching algorithms identify peptide variants from the reference spectra. 63 A set of variants is the proteomic profi le.
Epigenetic Profiles
Epigenetic changes occur as a result of environmental events, such that a putative epigenetic profile is unique to each individual because no two individuals will have the same environmental exposures.
Epigenetic alterations change in the absence of cell division or DNA sequence alterations.
Epigenetic differences due to environmental exposures add an additional level of distinction among individuals.
Epigenetic markers can also be used to identify body fluids (e.g., saliva, semen, blood).