DNA Fingerprinting: Principle, Steps, and Applications

The genome is the set of genes present in a cell. About 99.9% of the human genome is almost similar to each individual. But 0.1% of the variance in sequence defines an individual’s identity, making them different from each other.

The genome consists of both coding and non-coding region. The non-coding region is the repeated sequences that are inherited and do not code for the protein but make up most of the genetic DNA (deoxyribonucleic acid), whereas the coding region codes for proteins with a unique function.

Some of these non-coding regions contain repeated sequences that vary in length between individuals. These repeats are used to tell people apart in techniques such as DNA fingerprinting, DNA profiling, and DNA typing.

Figure: Bands Observed in DNA fingerprinting

Principle of DNA Fingerprinting

Almost all of the human genome is the same from person to person. The differences that make each person unique sit mostly in the non-coding DNA, in short sequences that are repeated over and over, one copy after another. These are called tandem repeats.

The key fact is this: the number of times a sequence repeats at a given spot varies from person to person. One person might have the block "GATA" repeated 7 times at a certain location. Another might have it repeated 12 times at the same location. Because each person inherits one version from each parent, everyone carries a combination of repeat numbers that is, across enough locations, effectively unique.

This is why the method works. If you measure the repeat number at one location, many people will share it by chance. But if you measure it at ten or more independent locations, the odds that two unrelated people match at every one become vanishingly small. The pattern becomes a fingerprint.

How does a repeat number turn into something you can see? Length. More repeats means a longer piece of DNA at that spot. When you separate DNA pieces by size on a gel, a person with more repeats gives a band that sits higher (a longer fragment), and a person with fewer repeats gives a band that sits lower (a shorter fragment). Reading the ladder of band positions is reading the repeat numbers, and that is the fingerprint.

Two properties make this useful in real cases:

1. It is inherited. A child's bands are a mix of the mother's and the father's. Every band in the child must be found in one parent or the other. This is what makes paternity testing possible.
2. It is individual-specific. Across enough locations, no two people (except identical twins) share the whole pattern. This is what makes forensic identification possible.

The repeat sequences used fall into two main groups, which differ in the size of the repeating unit.

• Minisatellites, also called VNTRs (variable number tandem repeats), have a repeat unit of about 10 to 60 base pairs. These were the original targets of DNA fingerprinting.
• Microsatellites, also called STRs (short tandem repeats), have a much smaller repeat unit of 1 to 6 base pairs. Modern DNA profiling uses STRs, because they can be copied easily by PCR and need only a tiny amount of DNA.

Introduction and History

DNA fingerprinting was discovered by Alec Jeffreys at the University of Leicester in September 1984. He was studying the human myoglobin gene when he noticed a short, repeated sequence inside it, a minisatellite. He made a probe from that repeated core sequence and used it on a Southern blot that happened to contain DNA from one of his lab technicians and both of her parents. The result was a smudgy pattern of bands that looked, in his own description, like a fuzzy bar code. Two things were immediately clear. The pattern was different for each person, and the child's bands were inherited from the two parents. He had found a way to identify any individual from their DNA.

The method proved itself quickly in the real world. In 1985 it settled a UK immigration case by proving a family relationship. Then, in the Colin Pitchfork case of 1986 to 1987, it was used in a criminal investigation for the first time. Two teenage girls had been murdered. DNA fingerprinting first cleared a man who had confessed but whose DNA did not match, and then identified the true killer from a mass screening. It is a defining example of the technique doing two things at once: freeing the innocent and convicting the guilty.

The reason the method works comes back to the repeat sequences described above. Because the number of repeats varies between people and is inherited from both parents, the band pattern is both individual-specific and traceable through families.

DNA fingerprinting uses segments of the genome made up of tandem repeats of non-coding nucleotide sequences known as satellite DNA. Tandem repetitions are sets of copies of the same nucleotide sequence and may repeat once or more. For instance, the tandem repeat CG CG CG CG CG repeats the sequence CG several times.

DNA fingerprinting targets these tandem repeats in the non-coding DNA, sometimes called satellite DNA. Beyond the minisatellites and microsatellites described above, a third and largest class exists: macro-satellites, with repeated units several kilobases long. D4Z4 and DXZ4 are examples.

A child inherits one set of repeat numbers from the mother and one from the father. This mix of inherited repeat lengths is what makes each person's pattern distinct, and it is the basis of DNA fingerprinting and genetic mapping.

Figure 1: Satellites with tandem repeats. Source: (Saeed et al., 2016)

Steps of DNA Fingerprinting

The process of DNA fingerprinting begins with the collection of samples, followed by DNA extraction which is then amplified and observed by electrophoresis or under X-ray.

Figure: Variable Number of Tandem Repeats Source: MGA2 09-06

Collection of specimen: The DNA sample is collected from body fluids such as blood, saliva, sweat, tears, tissues, and hair.

Extraction of DNA: DNA is extracted either by manual method or commercially available extraction kits. Two different methods further analyze the extracted DNA; polymerase chain reaction and restriction fragment length polymorphism.

Polymerase Chain Reaction (PCR) is a reaction that doubles the DNA copies in each cycle.

• After DNA isolation, the extracted DNA is subjected to PCR to obtain the number of copies of targeted sequences.
• Mathematically, the number of copies obtained can be calculated as, The number of copies = 2n, where “n” refers to the number of cycles. Over 20 cycles, one starting molecule yields about 1,048,576 copies (2 to the power of 20).
• The main objective of performing PCR is to amplify the small amount of targeted DNA sequence. Agarose Gel Electrophoresis can help visualize the amplified DNA.

In the Restriction Fragment Length Polymorphism (RFLP) route, genomic DNA is cut with a restriction enzyme, and the fragments are separated by size on a gel. The fragments are transferred to a membrane and detected with a labeled probe that binds the variable repeat regions. This route needs a relatively large amount of good-quality DNA. The restriction-enzyme step itself is described in detail in the separate article on Restriction Fragment Length Polymorphism.

Next, the nylon membrane is washed to remove the excess probe. The nylon membrane is then exposed to X-ray to ensure targeted DNA’s bound with a probe.

The positive sample shows dark bands when exposed to X-ray, whereas the negative sample shows no bands with respect to the ladder. This process is also known as autoradiography. Lastly, the dark bands in the membrane (DNA fingerprint) are compared with the known samples.

Reading the result.

The final pattern is a set of bands, one ladder of bands per sample lane. To interpret it, you compare lanes side by side. In a forensic case, the suspect's lane must match the crime-scene lane band for band to be considered a match. In a paternity case, every band in the child's lane must appear in either the mother's lane or the alleged father's lane. A band in the child that is present in neither parent excludes that man as the father. The strength of a match is then expressed as a probability, because a full match across many independent locations is extremely unlikely to occur by chance in unrelated people.

Figure: Restriction Fragment Length Polymorphism Source:GeeksforGreeks

Applications of DNA Fingerprinting

DNA fingerprinting is a widely used molecular technique with wide applications in different fields. Forensic analysis and determination of ancestral origin are the main areas of its applications. Following are some of the applications of DNA fingerprinting:

• To study evolutionary relationships by comparing repeat patterns between individuals and groups.
• To confirm paternity and to trace relationships in pedigree analysis.
• The criminal involved in the crime is easily investigated after matching the DNA from the crime scene with the suspects.
• DNA fingerprinting is also used in donor identification during an organ transplant.
• It is important in genetic counseling on genetic diseases and disorders.

How to Remember

The whole idea: a stutter you can count. A tandem repeat is DNA that "stutters," repeating the same short sequence again and again. Different people stutter a different number of times at the same spot. Count the stutters at enough spots and you have named the person. More stutters means more length means a higher band.

Mini vs micro by the numbers. Minisatellite has the bigger repeat unit (10 to 60 bp) and came first (Jeffreys, 1984). Microsatellite (STR) has the tiny repeat unit (1 to 6 bp) and is what modern profiling uses because PCR loves small targets. Small unit, small "micro."

Paternity rule in one line. Every band in the child must come from one parent or the other. A child band that matches neither parent rules that parent out.

The Pitchfork case, for why it matters. The first criminal use of DNA fingerprinting both freed an innocent man and caught the real murderer. The technique's power is that it can exclude as firmly as it can identify.

Key exam facts in one table

Where Students Get Confused

"What actually differs between people, the sequence or the number of repeats?" The number of repeats. Two people can have the exact same repeat sequence (say, GATA) at a location but a different number of copies of it. That difference in count is what changes the fragment length and produces a different band. DNA fingerprinting reads repeat number, not the letters of a gene.

"Minisatellite, microsatellite, VNTR, STR: how do these map?" Minisatellite and VNTR are the same thing, with a repeat unit of about 10 to 60 bp. Microsatellite and STR are the same thing, with a repeat unit of 1 to 6 bp. VNTR is sometimes used as a broad term for both. The simple rule: mini and micro refer to the size of the repeating unit, not the number of repeats.

"Why does more repeats give a higher band and not a lower one?" Because more repeats means a physically longer piece of DNA. Longer pieces move more slowly through the gel and stay nearer the top, so they appear as higher bands. Fewer repeats means a shorter, faster piece that travels further down.

"Is DNA fingerprinting the same as RFLP?" No. RFLP is one method that can produce a DNA fingerprint, by cutting DNA with a restriction enzyme and detecting variable fragments with a probe. DNA fingerprinting is the goal (identifying an individual), and it can be reached by RFLP or, more commonly today, by PCR-based STR typing. The restriction-enzyme mechanism itself is covered separately in the article on Restriction Fragment Length Polymorphism.

"Can it tell identical twins apart?" No. Identical twins share the same repeat pattern, so standard DNA fingerprinting cannot distinguish them. This is the one routine exception to individual specificity.

References

• Jeffreys A.J., Wilson V., Thein S.L. (1985). Hypervariable "minisatellite" regions in human DNA. Nature, 314(6006), 67–73. https://doi.org/10.1038/314067a0
• Jeffreys A.J., Wilson V., Thein S.L. (1985). Individual-specific "fingerprints" of human DNA. Nature, 316(6023), 76–79. https://doi.org/10.1038/316076a0
• Gill P., Jeffreys A.J., Werrett D.J. (1985). Forensic application of DNA "fingerprints." Nature, 318(6046), 577–579. https://doi.org/10.1038/318577a0
• Saad R. (2005). Discovery, development, and current applications of DNA identity testing. Baylor University Medical Center Proceedings, 18(2), 130–133. https://doi.org/10.1080/08998280.2005.11928051
• Saeed A.F., Wang R., Wang S. (2016). Microsatellites in pursuit of microbial genome evolution. Frontiers in Microbiology, 6, Article 1462. https://doi.org/10.3389/fmicb.2015.01462
• National Human Genome Research Institute. DNA Fingerprinting. Genome.gov Talking Glossary of Genomic and Genetic Terms. https://www.genome.gov/genetics-glossary/DNA-Fingerprinting