Genetic fingerprinting: a look inside
Submitted by minh on 22 February 2012
Even when we analyse all three blood group systems simultaneously, however, about one in ten people give identical results; this is what makes blood transfusions possible. For forensic purposes, however, it is a disadvantage: the results may tell you that your blood sample does not come from Suspect X, but they cannot tell you with any acceptable level of certainty that it does come from Suspect Y.
Advances were made in the 1970s and 80s, with the analysis of different forms of enzymes (isoenzymes) in red blood cells and blood serum. The certainty that the sample really came from the suspect depended on the number of proteins analysed (usually four); we call this certainty the power of discrimination. The power of discrimination that these combined techniques offered was still only 1:1000, better than the 1:10 power of blood group analysis, but still not good enough. To get a better power, we needed to take a closer look at our genetic composition.
Our genetic composition – who we are
Different people can have different numbers of repetitions of these sequences: one person may have five repeats at a specific DNA locus (site); another person may have seven. Using samples, e.g. from blood or semen, we can analyse the repetitive sequences at several DNA loci; we call this analysis a genetic fingerprint. Like fingerprints, genetic fingerprints can be used to distinguish individuals.
Although the term ‘genetic fingerprinting’ (or genetic profiling) is commonly used, not everybody is aware that it actually encompasses two very different techniques, only one of which is commonly used in forensics today.
Early genetic fingerprinting: restriction fragment length polymorphism
This means that if you cut the genome with a particular restriction enzyme, you will get around 730 000 restriction fragments of varying lengths. And this is where the VNTRs become important: the number of repeats in a particular VNTR cluster may vary between individuals, which means that the length of the corresponding restriction fragment will vary between individuals too (Figure 1). We call this phenomenon restriction fragment length polymorphism (RFLP).
Figure 1: Overview about two VNTRs from two different individuals. Sites cut by restriction enzymes (molecular scissors) are indicated by an arrow. Depending on the number of VNTR repeats, DNA fragments of different sizes are generated (see also Figure 2, Step 4)
Image courtesy of Sara Müller
Of the 730 000 restriction fragments, only some will differ between individuals – too few to be detected by eye. Instead, scientists used a technique called Southern blotting, which allowed only the sequences of interest to be visualised. To do this, they separated the restriction fragments according to size by gel electrophoresis, using an electric current to pull the charged molecules of DNA through a gel. The distance travelled was determined by the fragment size (Figure 2, Step 1). Next, they transferred the DNA to a membrane (Figure 2, Step 2) and applied a radioactively labelled probe that was complementary to the VNTR(s) of interest. The probe hybridised (stuck) to the matching sequences (Figure 2, Step 3) and by placing the membrane on an X-ray film, the scientists got a picture of the radioactively labelled bands, each of which represented a different length of fragment (Figure 2, Step 4). This picture was the genetic fingerprint.
So how many VNTRs needed to be compared to reliably distinguish between individuals? If the scientists chose VNTRs with enough variation (e.g. D1S80, which may be repeated anything from 15 to 41 times), they only needed to compare four different VNTRs to have a power of discrimination of 1:1 million – much better than the 1:10 offered by ABO blood typing.
Figure 2: RFLP analysis after digesting the DNA with restriction enzymes. Click on image to enlarge
1. Size separation of digested DNA fragments by gel electrophoresis. Large molecules with slow mobility in the gel can be seen at the top of the picture; small molecules with higher mobility in the gel are at the bottom
2.-3. Southern blot technique. Separated DNA is transferred to a membrane and subsequently detected on the membrane with radioactively labelled probes against VNTRs A and B
4. The exposed X-ray film shows an individual fingerprint for each person (compare to Figure 1)
Image courtesy of Sara Müller
The current technique: PCR-based genetic fingerprinting
For PCR analysis, we need STRs flanked by sequences that are identical in all human beings (we say these sequences are conserved). We then use primers – short molecules that are complementary to the conserved flanking sequences (genes 1134 and 1135 in Figure 4) – to initiate the PCR. Once the DNA has been amplified, we can separate it either by gel electrophoresis (Figure 5) or, in modern forensic science, by electrophoretic automated sequencing (Figure 6), and visualise it as a genetic fingerprint.
Figure 4: Schematic view of the STR D1S80 (the nomenclature ‘D1S80’ tells us that the STR is on chromosome 1, in region 80) from two individuals. The black arrows represent the primers used to amplify that specific STR. Click on image to enlarge
Image courtesy of Sara Müller
We have two copies of each chromosome, so we also have two copies of each STR. If, for each copy of the STR, someone has the same number of repetitions (i.e. the same allele), the PCR analysis reveals only one size of DNA fragment: the person is homozygous for that STR allele (green arrow in Figure 5, corresponding to individual 2 in Figure 4). If the two chromosomes carry non-identical alleles for that STR, we see two sizes of fragment and say that the person is heterozygous (red arrow in Figure 5, corresponding to individual 1 from Figure 4).
Figure 5: Genetic fingerprint of the D1S80 locus generated by school students (channels numbered 1–16) with their own DNA. The lane on the far right, labelled M, contains DNA fragments of known sizes, used as markers.
The individual indicated with the green arrow is homozygous for the D1S80 locus (only one band is visible). The individual marked by the red arrow is heterozygous (two bands). The blue arrows indicate two students who are heterozygous and have the same number of repeats for each allele at the D1S80 locus; this means that they cannot be distinguished by the fingerprint. They may be twins, but it is also likely that two unrelated persons will have the same number of repeats if only one STR is analysed
Image courtesy of Sara Müller
If we only analyse one STR, the chance of two unrelated people having the same PCR-based genetic fingerprint is high – between 1:2 and 1:100 (blue arrows in Figure 5). This is because STRs have fewer alleles and lower heterozygosity than the VNTRs used in RFLP-based genetic fingerprints. To overcome this disadvantage, we analyse multiple STRs simultaneously; with 16 STRs, as is common in forensic casework in Germany, we can achieve a power of discrimination of 1:10 billion (equivalent to one person in the world’s population; Figure 6).
Figure 6: Electropherogram of a woman, generated by multiplex PCR and subsequent electrophoretic automated sequencing. Eight STRs (D3, TH01, D21, D18, SE33, vWA, D8 and FGA) and amelogenin (which indicates the sex) were analysed. The blue, green and black curves represent amplified STRs (with repeat numbers below the peaks). The red curve is the marker (DNA fragment size labelled in bp). Click on image to enlarge
Image courtesy of Sara Müller
After Butler (2010)
Applications of genetic fingerprinting
And to complicate international forensic investigations, although there is a European recommendation to analyse 16 STRs, each country can decide which STRs to analyse, which makes comparisons difficult.
Genetic fingerprinting at school
The isolation of DNA at school gives the students a ‘wow’ moment when they realise that they are looking at the complete genetic information coding for an organism – a few cotton wool-like strands of DNA that were precipitated by alcohol. It is easy to perform at school using salivaw6 (or epithelial cells from commercially available kits), peas (Madden, 2006), tomatoes, onionsw7 or calf thymus (although check local restrictions on using calf thymus at school)w8.
If this equipment is not available, there are kits that both simulate and simplify the whole process of genetic fingerprintingw10. These kits contain fragments of DNA that simulate the amplification of different alleles of a single STR or VNTR. (In fact, they are restriction fragments of DNA from plasmid or lambda phage DNA.) The DNA requires electrophoresis and subsequent staining so that students are able to compare ‘amplified’ DNA sequences from a sample of evidence with those of several suspects. Of course, this is very different from detecting amplified STRs using electrophoretic automatic sequencing (and does not even accurately represent the visualisation of VNTRs using Southern blotting, as the DNA is stained directly on the gel), but it nonetheless demonstrates the principles of the analytical process. When using these simulation kits, the students should be made aware that the experiments give the impression that differences between individuals can be easily identified, which is not the case.
The authors would like to thank Wolfgang Nellen for his ideas about the article and for allowing the Science Bridge instructions to be made available free of charge.
They are also grateful to Shelley Goodman for her advice on using commercial kits at school.
Madden D (2006) Discovering DNA. Science in School 1: 34-36. www.scienceinschool.org/2006/issue1/discoveringdna
w1 – In 1930, Karl Landsteiner was awarded the Nobel Prize in Physiology or Medicine for his discovery of human blood groups. To learn more, see the Nobel Prize website (www.nobelprize.org) or use the direct link: http://tinyurl.com/7zjg2mw
w2 – To learn more about Alec Jeffreys’ discovery, see: www2.le.ac.uk/departments/genetics/jeffreys
w3 – The 1993 Nobel Prize in Chemistry was won by Kary B Mullis for his invention of the polymerase chain reaction (PCR). To learn more, see the Nobel Prize website (www.nobelprize.org) or use the direct link: http://tinyurl.com/7fkh7ku
w4 – To learn more about PCR, watch this video: www.youtube.com/watch?v=_YgXcJ4n-kQ
w6 – For instructions (in both English and German) for PCR-based genetic fingerprinting at school, see the Science Bridge website (www.sciencebridge.net) or use the direct link: http://tinyurl.com/89u7m53
w9 – For examples of commercial kits that can be used for PCR analysis at school, see the ‘crime scene investigator PCR basics kit’ on the Biorad website (www.bio-rad.com) and the PCR advanced kits on the Edvotek website (www.edvotek.co.uk).
w10 – For examples of commercial school kits that simulate and simplify the process of genetic fingerprinting, see the ‘forensic DNA fingerprinting kit’ on the Biorad website (www.bio-rad.com) and the ‘DNA fingerprinting by restriction fragmentation patterns kit’ (under ‘forensics’ and ‘DNA’) on the Edvotek website (www.edvotek.co.uk).
To learn more about repetitive DNA and methods (RFLP and PCR), see:
To find out more about PCR and STRs, as well as worldwide DNA databases and forensic case work, see:
For a classroom game on DNA detection, see:
The website of the DNA Initiative: Advancing Criminal Justice Through DNA Technology offers a free online course in forensic DNA analysis. Aimed at lawyers, it and the accompanying case study provide an excellent introduction to the topic. See: www.dna.gov/training/otc
To investigate a real database of STRs, visit the website of the US National Institute of Standards and Technology: www.cstl.nist.gov/biotech/strbase
To learn more about how genetic diseases are diagnosed, see:
If you enjoyed this article, you may like to browse the other biology-related articles in Science in School. See: www.scienceinschool.org/biology
Sara Müller studied biology, chemistry and education at the University of Kassel, Germany, and received her teaching degree for secondary schools in 2008. In December 2011, she finished her PhD thesis in the field of epigenetics, also at the University of Kassel. Since February 2012, she has been doing her practical training as a teacher in Göttingen, Germany. She has been a member of the executive board of Science Bridgew6 for the past seven years.
Heike Göllner-Heibült is a DNA forensic science expert with a background in molecular biology. She studied human biology at the Philipps University of Marburg, Germany, spending several months at the Erasmus University of Rotterdam, the Netherlands, and at the University of Cambridge, UK. In 2002, she finished her PhD in molecular biology at the Institute of Molecular Biology and Tumor Research in Marburg and started work in DNA forensics as a DNA expert and reporting officer. She currently works for the Forensic Science Institute of the office of criminal investigation in Berlin, Germany.
The idea of using DNA to identify one individual in the world is mind-blowing. The genetic fingerprinting of an individual relies on the sequences are that not used in coding, but how are these fingerprints created? This article explains.
Students may wish to discuss how genetic fingerprinting can help diagnose genetic diseases, as well as its application in the fight against poaching and species extinction. Ideally they should be able to try the technique for themselves, either as a real or a simulated experiment.
Shelley Goodman, UK