What you'll learn
- Why non-coding repeated DNA sequences are useful for identifying individuals.
- How PCR amplifies tiny amounts of DNA.
- How gel electrophoresis separates DNA fragments by length.
- How to interpret DNA profiles in forensic and relationship-testing contexts.
The starting point: DNA varies between people
Most human DNA is the same from person to person, but some regions vary a lot. DNA profiling focuses on these variable regions rather than sequencing a whole genome.
A particularly useful type is a short tandem repeat, or STR: a short sequence of bases repeated many times in a row. For example, one person might have 8 repeats at a particular position, while another might have 12.
Locus
A locus is the position of a gene or DNA sequence on a chromosome. At an STR locus, different people may have different numbers of repeats, producing DNA fragments of different lengths.
Because humans are diploid, you usually have two copies of each autosomal locus: one inherited from your biological mother and one from your biological father. These different versions are called alleles.
Why repeats are useful
STRs are useful in DNA profiling because the number of repeats varies between individuals, so the length of the DNA fragment at that locus can differ between people.
What is a DNA profile?
A DNA profile is a pattern of DNA fragments from several variable loci. In exams, you often see this as a pattern of bands on a gel, or as peaks in a graph from capillary electrophoresis.
DNA profiling
DNA profiling is the process of analysing DNA from an individual to produce a pattern that can be compared with other DNA samples, for example in forensic investigations or parentage testing.
DNA profiling is used to:
- compare crime-scene DNA with suspect DNA
- identify human remains
- test biological relationships, such as parentage
- compare individuals in conservation or breeding programmes
A DNA profile is not the whole genome
A DNA profile usually compares selected variable loci, not every base in the genome. A match means the tested loci match; it does not mean every part of the genome has been sequenced.
The overall workflow
A typical DNA profiling process involves:
- Collecting cells from a source such as blood, saliva, hair root tissue, semen or cheek cells.
- Extracting DNA from those cells.
- Amplifying selected STR loci using PCR.
- Separating the DNA fragments by length using gel electrophoresis or capillary electrophoresis.
- Comparing the pattern of bands or peaks between samples.
In older-style profiling methods, DNA may also be cut using restriction enzymes, which are enzymes that cut DNA at specific base sequences. Modern STR profiling commonly uses PCR primers that flank the repeat regions.
PCR: making many copies of a target DNA sequence
Crime-scene samples may contain only a tiny amount of DNA. Polymerase chain reaction, usually shortened to PCR, is a laboratory technique used to amplify a specific DNA sequence.
PCR
PCR is a technique that uses repeated temperature cycles to make many copies of a specific DNA region in vitro, meaning outside a living organism.
PCR needs:
- a DNA template, which is the original DNA containing the target sequence
- two primers, which are short single-stranded DNA sequences that bind either side of the target region
- free DNA nucleotides
- a heat-stable DNA polymerase, such as Taq polymerase
- a buffer containing suitable ions and maintaining pH
The same three stages repeat for many cycles.
Stage 1: denaturation
The reaction is heated to about 95 °C. This breaks the hydrogen bonds between complementary bases, separating the double-stranded DNA into two single strands.
Stage 2: annealing
The reaction is cooled to about 50–65 °C. Primers bind to complementary base sequences on the single-stranded DNA.
Stage 3: extension
The temperature is raised to about 72 °C. Heat-stable DNA polymerase adds free DNA nucleotides to the primers, forming new complementary strands.
Remember PCR temperatures
Think hot, cool, warm: hot to separate strands, cool so primers bind, warm so polymerase extends.
PCR amplification is exponential
In ideal conditions, the amount of target DNA approximately doubles each cycle. If you start with N0N_0N0 target DNA molecules and run nnn successful cycles:
N=N0×2nN = N_0 \times 2^nN=N0×2nwhere NNN is the final number of target DNA molecules.
Calculating PCR copy number
A sample contains 12 copies of a target STR region. Estimate the number of copies after 28 ideal PCR cycles.
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Use the PCR amplification model: N=N0×2nN = N_0 \times 2^nN=N0×2n.
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Substitute the starting number and number of cycles:
N=12×228N = 12 \times 2^{28}N=12×228. -
Calculate the power term:
228=2684354562^{28} = 268435456228=268435456. -
Multiply by the starting number:
N=12×268435456=3221225472N = 12 \times 268435456 = 3221225472N=12×268435456=3221225472. -
Give the answer in standard form: about 3.2×1093.2 \times 10^93.2×109 copies.
PCR is not perfectly exponential forever
The doubling model is an idealisation. In real PCR, amplification slows as primers, nucleotides or polymerase activity become limiting, and products may re-anneal to each other.
Controls in PCR
Good practical work needs controls.
A negative control contains all PCR reagents except template DNA. It should produce no DNA band. If it does produce a band, contamination is likely.
A positive control contains DNA known to amplify successfully. It checks that the PCR reagents and temperature cycle worked.
Why contamination matters
PCR is extremely sensitive, so even tiny amounts of contaminating DNA can be amplified and give misleading results.
Gel electrophoresis: separating DNA fragments
After PCR, the STR fragments need to be separated so their lengths can be compared.
Gel electrophoresis
Gel electrophoresis is a technique that separates charged molecules by applying an electric field across a gel.
DNA has a negatively charged phosphate backbone. When placed in an electric field, DNA fragments move towards the positive electrode.
The gel, often made from agarose, acts like a molecular sieve. Smaller DNA fragments move through the pores more easily, so they travel further than larger fragments in the same time.
A DNA ladder is run alongside the samples. It contains fragments of known lengths, usually measured in base pairs, abbreviated to bp. The ladder lets you estimate the sizes of unknown fragments.
Direction of movement
DNA is negative, so it moves to the positive electrode. The wells are placed near the negative electrode so fragments travel through the gel.
Band number is not enough
Do not just compare the number of bands. A match requires bands to be in the same positions, meaning the fragments are the same lengths.
Reading a DNA profile
Each band represents DNA fragments of a particular length. If two samples have bands in the same positions, those fragments are the same length at the tested loci.
For a strong identification, scientists compare many STR loci. The more loci that match, the lower the probability that the match happened by chance.
Identifying a matching DNA profile
A crime-scene sample has bands at 180 bp, 260 bp, 420 bp and 700 bp. Suspect A has bands at 180 bp, 260 bp, 500 bp and 700 bp. Suspect B has bands at 180 bp, 260 bp, 420 bp and 700 bp.
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Compare the crime-scene profile with Suspect A: the 180 bp, 260 bp and 700 bp bands match, but the crime-scene sample has 420 bp while Suspect A has 500 bp.
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Compare the crime-scene profile with Suspect B: all four band positions match exactly.
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Conclude that Suspect B matches the crime-scene DNA profile at the loci tested, while Suspect A does not.
DNA profiling and biological relationships
In parentage testing, a child’s alleles must be inherited from their biological parents. For each tested locus, one allele should match the mother and the other should match the father, unless there is a mutation or experimental error.
This is why DNA profiling can show relatedness as well as identity. Close relatives share more alleles than unrelated individuals.
A match is not automatically guilt
In forensic cases, a DNA match shows that DNA from that person may be present. It does not by itself prove when or how the DNA got there, or that the person committed a crime.
Practical reliability and evaluation
When evaluating DNA profiling evidence, think about:
- sample quality: old or degraded DNA may break into smaller fragments
- sample quantity: very small samples are harder to analyse reliably
- contamination: DNA from investigators or other samples may be amplified
- number of loci tested: more matching loci gives stronger evidence
- mixed samples: DNA from several people can make profiles harder to interpret
- chain of custody: samples must be labelled, stored and handled securely
In the exam
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When describing PCR, include the three stages in order: denaturation, annealing and extension, with the role of primers and DNA polymerase.
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When interpreting gels, compare band positions, not just the number of bands.
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When evaluating forensic evidence, mention contamination, sample degradation and the fact that a DNA match is evidence of presence, not proof of guilt.
Check yourself
- Why do smaller DNA fragments travel further than larger fragments in gel electrophoresis?
- What would it suggest if a negative PCR control produced a DNA band?
- In parentage testing, why should a child share alleles with both biological parents?
