Chromosomes, Genes and the Human Genome

If the DNA in a single human cell were stretched out, it would measure approximately two metres in length. That molecule must fit inside a nucleus roughly six micrometres across. The cell achieves this through a hierarchy of compaction, and understanding that hierarchy explains both why chromosomes exist as discrete structures and why forensic samples can contain intact chromosomal DNA even from dried biological material.

The first level of compaction is the nucleosome. A section of about 147 base pairs of DNA wraps roughly 1.65 times around a core of eight histone proteins (two each of H2A, H2B, H3, and H4). The result looks like beads on a string when viewed by electron microscopy. This structure reduces the effective length of the DNA by about seven-fold. A linker histone (H1) stabilises the connection between adjacent nucleosomes. At the next level, nucleosomes are coiled into a 30-nanometre chromatin fibre, achieving a further six-fold compaction. Further looping and scaffolding compress the fibre into the classic X-shaped chromosome visible during mitosis, where the total compaction factor reaches approximately 10,000-fold relative to the naked DNA.

This compaction is dynamic. During interphase (the non-dividing state of a cell), chromosomes are partially decondensed and genes are accessible to the transcription machinery. Regions that are being actively transcribed tend to be in more open (euchromatic) configurations; silenced regions are in tighter (heterochromatic) configurations. For forensic biology, the important point is that most cells in biological evidence, including dried bloodstains, epithelial cells in saliva, and spermatozoa, contain intact chromosomes in their nuclei, and the DNA within those chromosomes can be extracted and analysed by standard laboratory methods.

The Human Genome Project, completed in draft in 2001 and in finished sequence by 2003, established that the haploid human genome contains approximately 3.2 billion base pairs of DNA. The diploid genome in most somatic cells therefore contains about 6.4 billion base pairs. Distributed across 23 chromosomes, the chromosomes range in size from chromosome 1 (about 249 million base pairs) to the Y chromosome (about 57 million base pairs, much of which is repetitive heterochromatin).

Protein-coding genes number approximately 20,000, and their exons (the portions that are translated into protein) together account for roughly 1.5 percent of the genome sequence. The rest is composed of introns (non-coding intervening sequences within genes), regulatory elements (promoters, enhancers, silencers), non-coding RNA genes (including microRNAs and long non-coding RNAs), repetitive sequences, transposable elements, and sequences with no currently assigned function. This non-coding majority is not 'junk': much of it is transcribed, some of it regulates gene expression, and it is within this fraction that the highly polymorphic STR loci used in forensic profiling are found.

The 22 autosomes are numbered roughly by size (chromosome 1 being the largest), and each individual carries two copies, one inherited from each parent. The 23rd pair is the sex chromosomes: females carry two X chromosomes and males carry one X and one Y. This distinction has direct forensic relevance. Autosomal STR profiling uses markers on the 22 autosomes and can identify individuals regardless of sex. Y-chromosome STR profiling traces the paternal line. X-chromosome STR profiling follows inheritance rules intermediate between autosomal and Y-linked, and is particularly useful in kinship analysis.

A protein-coding gene has a defined structure. Moving along the DNA in the direction of transcription, a gene contains a promoter region (where transcription factors and RNA polymerase bind), a 5' untranslated region, one or more exons (coding sequences), introns separating the exons, a 3' untranslated region, and a polyadenylation signal. When the gene is expressed, the entire stretch from the 5' cap to the poly-A tail is transcribed as pre-mRNA; the introns are then spliced out to produce mature mRNA, which is exported from the nucleus and translated into protein.

Introns are non-coding sequences within genes, but they are not unimportant. Some introns contain regulatory elements; alternative splicing of introns can produce multiple protein variants from a single gene. The intronic fraction of the genome is larger than the exonic fraction, and intronic sequences are where many STR loci are located. An STR locus sitting inside an intron is not part of the mRNA; it has no effect on the protein product; and variation in the number of repeats does not alter the gene's function. This is why such loci are attractive for forensic profiling.

Beyond introns, the genome contains large intergenic regions, which are the stretches between genes. These regions include regulatory sequences that control gene expression over long distances, non-coding RNA genes, the centromeres (repetitive regions at the physical centre of each chromosome that are critical for cell division), and the telomeres (protective caps at chromosome ends). Repetitive elements, including satellite DNA, minisatellites, and microsatellites (another name for STRs), are distributed throughout. The density of STR loci is roughly one per every few thousand base pairs, giving the genome millions of potential STR sites, of which a carefully chosen subset is used in forensic panels.

No two people (other than identical monozygotic twins) have the same genomic sequence. The differences arise from several mechanisms. Single nucleotide polymorphisms (SNPs) are positions where a single base differs between individuals; there are roughly 4 to 5 million SNPs distinguishing any two unrelated people. Insertions and deletions (indels) occur when short stretches of sequence are present in some individuals but absent in others. Copy number variants (CNVs) are larger regions, sometimes encompassing whole genes, that are duplicated or deleted in some people. STRs vary in the number of times a short motif is repeated: a locus with the motif AGAT repeated 7 times in one person and 12 times in another illustrates allelic variation at that locus.

STR variation arises primarily through replication slippage. During DNA replication, the nascent strand can temporarily dissociate and re-anneal out of register on the template strand at a repeat region. This produces a loop in either the template or the nascent strand, resulting in the insertion or deletion of one or more repeat units in the daughter molecule. Because this happens with measurable frequency at STR loci, the number of repeats at a given STR site drifts over generations, generating the allelic diversity that makes these loci polymorphic.

Population genetics determines how useful a given locus is. If an allele is very common, finding it in crime-scene evidence and in a suspect tells us little, because many people in the population carry it. If an allele is rare, finding it in both samples is more informative. Forensic panels are designed to maximise the product of allele frequencies across multiple independent loci, producing a combined match probability that can be extremely small, often less than one in a billion for a full 20-locus CODIS profile.

Not every polymorphic locus in the genome is suitable for forensic profiling. Selection involves balancing several technical and ethical criteria. The Scientific Working Group for DNA Analysis Methods (SWGDAM) in the United States, the European DNA Profiling Group (EDNAP), and equivalent bodies in other jurisdictions developed the criteria that underpin current panels.

• High polymorphism: the locus must have many alleles in the population, with no single allele dominating. This is measured by heterozygosity. CODIS loci typically have heterozygosity values above 0.7.
• Non-coding location: the locus must not encode medical, phenotypic, or other sensitive personal information. Placing profiling loci in introns or intergenic regions achieves this.
• PCR amplifiability from degraded samples: the target region must be short enough to survive degradation and be amplified reliably. Most forensic STR amplicons are under 400 base pairs; newer panels use even shorter amplicons for degraded samples.
• Low mutation rate: the locus must be stable enough that parent-child allele sharing is reliable for kinship analysis, typically a mutation rate below 1 in 500 transmissions.
• Autosomal independence: loci on different chromosomes (or far apart on the same chromosome) assort independently at meiosis, so allele combinations can be treated as statistically independent, allowing the multiplication rule in profile frequency calculations.

The CODIS 20-locus panel, expanded from 13 loci in 2017, includes loci distributed across 15 different chromosomes. The European Standard Set of 17 loci, defined by the European Network of Forensic Science Institutes (ENFSI), overlaps substantially with CODIS and has been updated to align with the CODIS expansion. India's proposed panel under the DNA Technology Regulation Bill follows a similar design philosophy. Harmonisation of locus panels across jurisdictions matters for transnational investigations, allowing DNA profiles generated in one country to be compared with databases in another.

For a deeper view of how profiling fits into forensic biotechnology more broadly, see the Forensic Biotechnology subject, and for how profiling interacts with serological identification of biological fluids, see Forensic Serology.

The sex chromosomes, X and Y, follow different inheritance rules from the autosomes, and those differences produce specialised forensic tools. The Y chromosome is inherited almost entirely unchanged from father to son, because it undergoes very little recombination. A set of Y-STR markers therefore produces a haplotype that is shared by all males in a paternal lineage. This makes Y-STR profiling useful for tracing a male lineage in historical cases or for identifying a male contributor in a complex mixture containing predominantly female DNA, as in some sexual assault cases where vaginal cells swamp the male fraction.

The limitation of Y-STR profiling follows directly from the inheritance rule: a Y haplotype cannot distinguish between brothers, a father and his sons, or any other males in the same paternal lineage. A match between crime-scene Y-STR evidence and a suspect's Y haplotype implicates the lineage, not the individual. Courts in the United States, United Kingdom, and European jurisdictions have grappled with how to present Y-STR evidence to juries; the standard approach is to report a haplotype frequency in the relevant population database rather than a match probability.

Mitochondrial DNA (mtDNA) sits outside the nucleus, in the mitochondria, and is inherited maternally through the egg cytoplasm. Each cell contains hundreds to thousands of mitochondria, each with multiple copies of the circular 16,569-base-pair mtDNA genome. This copy number advantage makes mtDNA analysis valuable when nuclear DNA is absent or severely degraded: shed hairs without roots, ancient bones, and highly burnt remains may yield mtDNA when nuclear STR profiling fails. Like Y-STR, mtDNA sequence types are shared by matrilineal relatives and cannot distinguish between them. The forensic analysis of mtDNA focuses on the hypervariable regions (HV1 and HV2) of the control region, where sequence variation between individuals is greatest.