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Genetic fingerprinting and DNA profiling

Variable number tandem repeats (VNTRs)

These are fairly short, usually non-coding, sections of DNA, consisting of the same sequence of nucleotides, repeated in rows on chromosomes. The numbers of these repeats - sometimes known as 'stuttering' - are highly variable, and each section is joined to one or more identical copies without any intervening sections with different nucleotide sequences. Since there may be different numbers of repeats at the same location on the chromosome ('polymorphic loci'), they may be considered as multiple alleles.

They appear to have been produced by a random process ('slippage') during nuclear division and subsequently copied as chromosomes are replicated, so they are passed on from parents to offspring in the same way as ordinary alleles, and found in all the body cells. Consequently any of this material can be traced back to either the male or female parent .

There are two main classes, depending on the number of repeated nucleotides:

Minisatellites are generally 10-100 nucleotides in length.

Microsatellites - also known as short tandem repeats (STRs) - are much smaller - 1-6 nucleotides in length

Different individuals can have the same repeating sequences, but different numbers (or none) of them, i.e. the numbers vary but the sequences themselves do not. It is said that the probability of two individuals having the same (number of) VNTRs is very low, so this information can be used to identify a person based on samples of DNA from cells.

This has been found to have great significance in the evaluation of evidence in forensic science, as well as paternity and immigration disputes, and in the study of evolution.

Exceptions to the rule

In general, these extra sections of DNA are found in non-coding sections of the genome.

However, there are a number of trinucleotide repeats located in coding areas of the genome which are responsible for serious conditions, several inherited as dominant genes, although their symptoms may take some time to develop.

Huntington's disease is characterised by an accumulation of CAG repeats on chromosome 4. Less than 36 of these repeats has no real effect but greater numbers are associated with degenerative changes of the brain and nervous co-ordination which may typically appear between the ages of 30 and 50. Longer repeats lead to an earlier onset.

There are several forms of Spinocerebellar ataxia which cause degeneration in muscular control resulting from CAG repeats in other parts of the genome.

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disorder causing muscle cramps and progressive weakness due to degeneration of motor neurons in the brainstem and spinal cord, and it is associated with mutation of the androgen receptor (AR) gene, which is carried on the X-chromosome, so it is sex-linked. In fact males with less than 35 CAG repeats are unaffected but increased numbers result in increasing risk of development of SBMA.

Genes containing increased numbers of CAG repeats are collectively known as polyQ diseases.
Explain why they are given this name.
> The DNA triplet CAG (transcribed into the mRNA triplet GUC) causes the amino acid glutamine (Q) to be added to the developing polypeptide chain, so the resulting protein contains a number of glutamine residues.

Development of DNA fingerprinting

Alec Jeffreys with an autoradiogram showing a set of DNA minisatellite bands Sir-Alec-Jeffreys (36K)
Alec Jeffreys discovered and developed this process in 1984, at the University of Leicester.

He said that he stumbled across the background to the technique 'purely by accident'.

He was investigating the gene coding for myoglobin, the oxygen-storing protein in muscle tissue.

He hydrolysed a DNA sample using a restriction enzyme, and then attempted to anneal/hybridise the resulting DNA fragments to a radioactive DNA probe.

After gel electrophoresis and development of a photographic film he had left in contact with the gel, he was surprised to obtain an autoradiogram with a number of dark bands, rather like a bar code used to identify supermarket products.

The minisatellites causing these bands and their number were found to be extraordinarily variable between people, and their combinations represented differences at a unique level.

These differences were also found to be inherited like ordinary alleles.

The University of Leicester was at the forefront of the development of these techniques for a number of years. Professor Sir Alec Jeffreys has received a number of honours including Fellowship of the Royal Society (FRS) in 1986, a knighthood in 1994 and Companion of Honour in 2017.

What did each band on the autoradiograph represent?
> a section of DNA
> with a complementary sequence to the probe

Why are they spaced out on the autoradiogram?
> because they are different lengths (some of which is not complementary to the probe)
> shorter sections travel further/faster down the gel

Why are some of the bands on the autoradiogram darker than others?
> because they contain multiple repeats of the same sections of DNA bases which bind with the probe, or there are many copies of DNA sections of the same length

What is the significance of the large number of bands on the autoradiogram?
> This shows there are many identical copies of the same section of DNA - 'minisatellites'

A protein or polypeptide section corresponding to the DNA sequence of the probe has not been found.
Explain why.
> This DNA is 'non-coding' - in introns, not exons, so it is 'spliced out' of pre-mRNA after transcription.
The original 'Genetic Fingerprinting' technique involved Restriction Fragment Length Polymorphisms (RFLP) based on minisatellite VNTRs:
The drawback to the technique is the fact that (relatively) large amounts of well-preserved DNA are required.

Alec Jeffreys was also involved in the development of more advanced techniques which solved many of the problems with the early 'genetic fingerprinting' process.

Some questions about the process

What do restriction enzymes do?
> cut DNA into short sections at same sequence
Why are the same restriction enzymes used on samples?
> Samples to be compared must get same treatment And the enzymes must not cut in the minisatellite VNTR regions
How does electrophoresis work?
> charged molecules are drawn towards electrode - Phosphate groups are negatively charged, so the DNA moves towards the anode (positive electrode)
Why does hybridisation require heating (and cooling) ?
> to open out double helix and allow probe to bind to complementary sequence
What information does the position of bands give?
> Size of DNA fragments - shorter sections move further

DNA profiling

More recently identification methods using PCR and short tandem repeats (STRs) - microsatellites - have become standardised. This can be used with smaller samples, as targetted sections of the DNA are amplified by the technique. And shorter (usually 4-nucleotide) DNA oligonucleotides can be used as probes, so it is possible to get results from slightly degraded samples.

These probes - possibly labelled with different coloured dyes - bind to selected sites ('loci') on multiplex sections taken from chosen chromosomes, and automated chemical analysis using capillary electrophoresis generates a series of peaks (electropherograms) which are interpreted as a set of numbers based on the numbers of DNA repetitions ('alleles') at those sites. In fact these numbers are in pairs. This is because there are 2 sets of chromosomes in diploid organisms.

The combination of resulting numeric codes turn out to be very individual, and they have been used in a variety of forensic situations. There are a number of databases utilising DNA profile data around the world.

Separation of five markers on chosen chromosome loci

electropherogram (31K)
The number of repeats in the two alleles for each marker are shown underneath.
The fourth (single peak) shows that the individual was homozygous for this allele.

Vertical scale = RFU (relative fluorescence units)
Horizontal scale = distance along combined DNA templates (base pairs)
Credit: Kline/NIST

Use of genetic fingerprinting and DNA profiling

Determining genetic relationships

Each individual inherits half of their DNA from their father and half from their mother.

In their genetic fingerprints, this is reflected in the constituent bands produced from accumulated minisatellites .
A child should show half of the bands on their autoradiograph corresponding with (at the same level as) those of their father and half with those of their mother but it is essential that there are no mismatches.

And with STR profiling of microsatellites, the child should receive either one or the other number of DNA repetitions (alleles) at each locus corresponding to each parent.

This process can be used to confirm or deny paternity, in cases under question.

It can also be used to confirm family relationships, in immigration disputes.

Furthermore, it can also be used to correlate with previous generations, with grandparents showing a 25% match of bands or repetitions.

Determining genetic variability within a population

DNA profiles can be compared to determine genetic relationships within different populations of animals or plants by looking for similarities in the banding pattern or numbers of repeats.

This could be applied in situations where inbreeding is to be particularly avoided: within zoos and in the maintenance of rare breeds

Small section of an autoradiogram showing members of a family
family_autoradiogram (6K)

Inheritance of alleles at different STR loci
Locus Father Mother Child
D3S1358 16, 17 18, 18 17, 18
TH01 7, 9 9, 9 9, 9
D21S11 29, 32.2 30, 31 31, 32.2*
D18S51 14, 17 13, 19 17, 19
D5S818 11, 13 11, 10 11, 11
D13S317 8, 11 8, 8 8, 8
D7S820 11, 12 10, 10 10, 11
D16S539 11, 12 10, 12 10, 11
CSF1PO 11, 11 12, 12 11, 12
vWA 17, 18 17, 19 18, 19
D8S1179 14, 15 15, 15 14, 15
TPOX 11, 11 11, 11 11, 11
FGA 23, 24 21, 23 23, 24
More about these loci below
* These seem to be variant versions of the alleles - the extra number signifying the number of extra bases

Application in forensic science

Crime scene analysis

In rape cases, semen samples can be taken, stored and used as evidence for comparison with DNA from accused suspects. If properly stored, these may be used at a much later date.

Other sources of genetic material may be collected in different potential crime scenes: blood, cigarette ends and drinking containers.

Identification of body remains

DNA may still be recoverable from bones or other body parts, and this may be compared with DNA from reliable sources, or family members.

Genetic databases

There are several databases containing DNA profiles maintained by different agencies around the world.

These concentrate on a number of gene loci spread amongst the human genome which have been shown to contain variable numbers of alleles - STR microsatellites. The proportions of each of these alleles in the general population have been determined, so that an estimate of the probability of matching two samples (such as crime scene vs suspect, or in a paternity dispute) can be calculated by multiplying all the individual allele freqencies.

Some countries maintain a number of versions of the database corresponding to different racial groups, in order to make allowances for differences in the distribution of different alleles, and the probability of making a match.

There are a number of supply companies producing kits to be used in these analyses and the details of their techniques (primers,etc) are considered commercial secrets.

Codis_profile (82K) Some loci are named after specific enzymes, others after the specific location on the chromosome (number after D in these cases)
Check out the NIST Standard Reference Database (link below).

Image courtesy PD-USGov-NIST
The American version of this - Combined DNA Index System (CODIS) - was set up with 13 loci but it has now been expanded to include 20 loci. Originally it focussed on those accused of sex offences but it has been extended to cover those accused of felonies. There is also a missing persons database.

The UK National Criminal Intelligence DNA Database (NDNAD) is based on 16 loci. It was set up in 1995 and has gone through a number of upgrades based on improvements in analytical techniques. In 2020 It contained 6.6 million profiles - approximately 10% of the population.

Anyone arrested in England and Wales on suspicion of involvement in any recordable offence will have had their DNA sample taken and profile data will be stored in the database, whether or not they are subsequently charged or convicted. However, those not charged or not found guilty must (or may?) have their DNA data deleted within a certain period of time.

Familal searching

If a person's DNA profile on a database is a close match to DNA from a crime scene, it is possible to infer that the real offender may in fact be a relative of theirs. In a number of cases this has led to successful prosecutions. In fact this connection can usually only be relied on in the context of parents and children, or between siblings, which share 50% of DNA. More distant relatives (cousins etc) become less useful as the degree of sharing of DNA becomes more dilute.

Other topics you may find interesting

Genealogical testing

A number of companies offer insights into your family history, as well as other factors such as susceptibility to various diseases, on the basis of DNA testing.

Surprisingly, some of this information has found its way into databases above!

Other related topics on this site

(also accessible from the drop-down menu above)

Gene expression
Recombinant DNA technology - see PCR
Identification and diagnosis of heritable conditions

Simpler treatment
Genetic Fingerprinting

Web references

Alec Jeffreys and Genetic Fingerprinting - some interesting stories about the development of this procedure

Forensic DNA Profiling and Database from a Malaysian perspective

Killer breakthrough - the day DNA evidence first nailed a murderer

DNA fingerprinting Some good detail from a website with an odd set of themes

DNA profiling From Wikipedia, the free encyclopedia

Short tandem repeat typing technologies used in human identity testing

Should we be making use of genetic genealogy to assist in solving crime? A report on the feasibility of such methods in the UK. The BFEG is an advisory non-departmental public body, sponsored by the Home Office that provides advice on ethical issues in the use of biometric and forensic identification techniques

Forensics, DNA Fingerprinting, and CODIS How ethical is it to keep a database of convicted felons' DNA profiles? Can we rely on DNA fingerprints for conviction? Many ethical issues surround the use of DNA in forensic technology.

Thirty years of DNA forensics: How DNA has revolutionized criminal investigations DNA profiling methods have become faster, more sensitive, and more user-friendly since the first murderer was caught with help from genetic evidence by Celia Henry Arnaud

Paternity testing and forensic DNA typing by multiplex STR analysis using ABI PRISM 310 Genetic Analyzer - A readable account of the background to using this apparatus in Egypt, with plenty of backup data, phanerograms and tables of allele data explaining the different contexts in which this was used.

NIST Announces Program to ID Human Cell Lines for Research - The National Institute of Standards and Technology (NIST) has announced that it is launching a project to collect and catalog DNA identification data for up to 1,500 human cell lines used in biological and medical research

Short Tandem Repeat DNA Internet DataBase - NIST Standard Reference Database SRD 130 - fascinating data about seqences etc

DNA-17 Profiling - Legal Guidance

Combined DNA Index System - From Wikipedia, the free encyclopedia

United Kingdom National DNA Database - From Wikipedia, the free encyclopedia

Richard III: Discovery and identification - A historical and scientific story that is very close to home, from the University of Leicester-

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