DNA PROFILING

Index

• What is DNA?
• Structure of DNA
• For what cases is DNA profiling used?
• Extraction of DNA
• DNA Profiling Techniques
• The use of PCR in DNA Profiling
• Detecting the variation
• History of DNA profiling in Victoria
• DNA profiling at present
• Trace DNA
• How are results interpreted?
• What do the DNA profiling results mean for a case?
  • Cost
  • Time and procedure
  • Admissibility in Court
• Checks and balances for civil liberties
• Biological implications of DNA profiling
• Some Statistics (Victoria)
• Glossary
• Links

The analysis of the DNA is known as DNA Profiling or DNA typing.  DNA fingerprinting is not the appropriate term.

What is DNA?

The initials stand for 'deoxyribonucleic acid', found in the cells of all living things, including the human body. The DNA is a very long molecule and is found in the nucleus of cells.

Although each person's DNA is unique (unless he/she has an identical twin), the techniques for identification only look at small parts of the DNA.

It is important to realise that a person will have the same DNA throughout their body in every cell with a nucleus. That is, the same DNA profiling results for one person will be obtained whether testing blood or semen or muscle tissue.

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Structure of DNA

The DNA molecule actually consists of two strands twisted about each other in the shape of a spiral staircase (double helix). The building blocks of the strands are referred to as bases. There are four different types of bases in the DNA molecule and it is the sequence of these that determines our inheritable characteristics. The bases from each strand bind to each other, holding the molecule together as in the stairs on a spiral staircase. As each type of base will only bind with another specific type, the two strands are said to be 'complementary'. Two bases binding together are referred to as a base pair (bp).

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For what cases is DNA profiling used?

It depends on many factors. DNA profiling is typically carried out when human biological fluid or tissue is found at a crime scene and used as evidence to link to, or exclude from, that scene, a possible suspect. Some examples are:

• A murder where it appeared that a struggle took place and blood from the murderer was left at the scene. This would be compared to a sample of blood from the suspect.
  
• A murder where a blunt instrument was used and a suspect was found with a club on which there were dried blood stains. The blood on the club would be compared to the victim's blood.

• A rape case where seminal fluid from the offender can be taken from the victim. This would be compared to a sample of blood from a suspect.

DNA profiling can only be used if there is enough DNA in the samples, and is only useful in comparing samples. Crime samples can be compared to a sample from a known suspect or compared to a database of DNA profiling results from other scenes or from convicted offenders. If there is a 'match', the two samples may have originated from the same person.

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Extraction of DNA

Before DNA can be profiled, it must be initially extracted from the samples.

The basic method of extraction used at VFSC is referred to as 'Chelex DNA extraction'. The sample is boiled with the Chelex. This breaks down the proteins and other cellular material. The Chelex prevents the DNA being broken down. The DNA is then separated from the remains of the proteins and other cellular material.

This method varies slightly depending on the tissue involved. Generally VFSC analyses blood, hair and semen. Hair and spermatazoa have particularly tough cell membranes, which require the addition of other chemicals to break them down.

Once the DNA is extracted the amount of DNA present in the sample is determined.

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DNA Profiling Techniques

There are two DNA profiling techniques used in forensic science laboratories throughout the world;

1.     Restriction Fragment Length Polymorphism (RFLP);
2.     Polymerase Chain Reaction (PCR)

The RFLP method was the first DNA profiling technique used in casework in Victoria in 1989. It involves cutting up the DNA into small fragments with molecular scissors known as restriction enzymes and determining their length. This is done by electrophoresis ie placing the fragments on a gel that acts as a molecular sieve. When a voltage is applied the larger fragments travel a lesser distance than the smaller ones because the gel molecules hinder the movement of the fragments.

The DNA fragments on the gel are then transferred to a special membrane to which is applied a 'probe', a radioactive piece of DNA that specifically attaches to certain fragments depending on their type .The position of the probe is found by placing the membrane next to a sheet of radioactive film, resulting in the DNA fragments appearing as 'bands'.

The discovery of the PCR technique in 1984 has had a huge impact on the field of molecular biology (the study of DNA). Its applications have revolutionised the methodology in all fields of genetics. The method involves repeatedly copying a small area of the DNA molecule.

PCR many advantages over the RFLP method. The main advantage being that it is more sensitive; i.e. it is able to be used with smaller amounts of DNA or damaged DNA. It is also less time consuming than RFLP analysis. For these reasons PCR was introduced for casework and is the technique currently in use.

The PCR reaction involves repeatedly amplifying (copying) a small region of the DNA molecule, so that at the end of the reaction that particular piece of DNA is present in greater (more detectable) amounts.

To carry out the reaction a machine called a thermal cycler is used, which subjects the DNA to repeated cycles of heating and cooling. The double-stranded DNA is heated and the separated strands are allowed to bind to 'primers'. These primers are pieces of DNA made so that they attach to the DNA at each end of the region to be copied. This single-stranded DNA is then copied to produce two new double stranded pieces of DNA. This heating/binding of primers and copying is repeated approximately 30 times, each time doubling the amount of DNA in the region of interest.

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The use of PCR in DNA Profiling

The regions of DNA that are used for DNA profiling are regions that show variation between individuals allowing them to be grouped into different types. The different areas of the DNA molecule are referred to as loci (this is the plural; the singular is locus). The loci are given names such as vWA, FGA, D1S80, which represent their position on the DNA molecule.

There are two types of variation:

a)    Sequence variation (e.g. HLA-DQA1)

The different types in these systems are distinguished from one another by variation in the order of subunits (bases) that make up the DNA in the particular region being examined.

b)    Length variation (e.g. D1S80, HUMTHO1, FES and vWA). The different types in these systems are distinguished from one another by variation in the number of repeats of a core subunit, within the region of interest i.e. a short sequence of bases is repeated over and over again - how many times it is repeated determines the type.
These areas of DNA are referred to as variable number tandem repeats (VNTR's)

If the core subunit is particularly small (1-5 bases long) the subunit is referred to as an STR (Short Tandem Repeat).  HUMTHO1, FES and vWA are examples of this.

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Detecting the variation

A detection system is required to determine the difference between DNA types once amplification has taken place. The detection system used depends on the type of variation i.e. sequence or length.

At present, only systems using length variation are routinely in use in casework. Using this method the variation is detected using electrophoresis. The millions of copies of DNA produced by the PCR reaction are placed on a gel in a line. An electrical voltage is applied to the gel and the DNA then travels through the gel because of electrical charge of the DNA molecules. The larger molecules travel a shorter distance through the gel than the smaller ones because the gel molecules hinder the movement of the DNA. The distance the DNA copies have travelled through the gel is compared to the distance that known standards have travelled.

The samples are positioned on the gel like this:

					direction of movement
_______	_______	_______	_______	_______
Victim	Suspect	Sample (from scene)	Husband	Standards (Numbers are number of repeats of the core sub-unit sequence

The results look like this:

LOCUS 'C' RESULTS

_______				______ 2
	_______	_______		______ 4
_______				______ 6	Direction
			_______	______ 8	of movement
	_______	_______		______ 10
			_______	______ 12
Victim	Suspect	Sample (from scene)	Husband	Standards (Numbers are number of repeats of the core sub-unit sequence

Conclusion:

The sample from the scene could not be from the victim or husband.

The suspect could be the source of the scene sample.

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History of DNA profiling in Victoria

RFLP

DNA Profiling was first introduced into Victorian casework in 1989 and was used either by itself or in conjunction with PCR until 1994.

HLA-DQA1

HLA-DQA1 was the first PCR technique used in casework in 1991. This system relies on sequence variation to differentiate between the different types.

Using this method the variation is detected using 'probes' which recognise only specific variations in the DNA sequence. The millions of copies of the DNA produced by the PCR reaction are washed over a membrane to which the various probes are bound. The probes only attach to the copies of DNA containing a particular DNA sequence.

A colour reaction is used to detect the presence of copies of DNA bound to the probes on the membrane.

The results looked like this:

The coloured dots indicate the DNA type.
The coloured 'C' dot indicates the test is working correctly.

Conclusion: This individual is HLA-DQA1 type 2,4

D1S80

D1S80, which is a PCR based VNTR system, was introduced for casework in 1993. The difference in types in this system is based on length variation. The core subunit, which was repeated, is 14-16 base pairs in length. It is the number of repeats of this subunit which determines an individual’s type.

Short Tandem Repeat (STR) systems

In 1994, the first of the STR systems was introduced for casework. HUMTHO1 was the first locus used, followed by FES and vWA. The STR systems have several advantages over the VNTR systems; the main ones being that they have a higher probability of producing a result if the DNA is damaged and they can be combined in a multiplexing system. (See next section for explanation.). An example of damaged DNA is the DNA in a crime scene sample which has started to deteriorate before it was collected. This can occur due to the action of bacteria, heat or other adverse environmental conditions.

Semi-Automated DNA profiling

In 1995, the ABI-Sequencer or 'Genescan' was introduced for casework. This machine semi-automates the profiling of the STR systems (FES, vWA, HUMTH01 and others). The DNA molecules produced in the PCR reaction are labelled with a fluorescent dye. The 'Genescan' machine determines what type the DNA molecule is at the different loci by detecting the movement of the fluorescence through the gel using a laser.

To perform the PCR reaction for a number of different loci in the one test tube, each loci being marked with a different coloured dye. This is referred to as multiplexing. The sample containing the copied DNA from all the loci is placed on the 'Genescan' and the types of a sample at all the DNA loci are determined by the machine recognising the different dyes as different loci.

The introduction of the 'Genescan' system and multiplexing means that many samples can be run at the one time and that a DNA profile with typings at many different loci can be obtained for the majority of sample with just one gel run.

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DNA profiling at present

At present, a system that allows us to amplify and type 9 STR loci in casework is used. It also allows the sex of a particular sample to be indicated using an area of DNA amelogenenin.

The use of so many loci increases the probability that two individuals will be distinguished, i.e. the more systems, the greater the evidential value.

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Trace DNA

A recent development discovered in Victoria is that a DNA profile can be obtained from objects touched by skin, providing a powerful new tool for crime scene investigation.

However, the high sensitivity of this method means that extra caution must be taken when exhibits are handled. If a particular exhibit is handled by a number of people the DNA profiling results indicate a mixture; so interpretation is not always straightforward.

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How are results interpreted?

For each locus a database containing the frequency each type which occurs in the population. This is used to determine at what frequency a particular combination of types is expected to occur in the population.

When the results of all the loci are known, they could be something like this.

The percentages can be multiplied together and statistically corrected.

The results could then be written as-

'The donor of the blood sample in the bag labelled 'J. BROWN' could not be excluded as the source of the biological material in the blood found at the scene.
This DNA evidence is 1 in 1 million (1,000,000) times more likely to have arisen if the scene sample came from 'J. BROWN' than if it came from a random member of the Caucasian population.
In my opinion, in the absence of evidence to the contrary, this provides strong support to the proposition that the samples have the same source.'

Not every case will give a result of 1 in 1 million. The number will vary, depending on how much scene sample there is, and therefore, how many typings can be done and on how common are the DNA types that were found: the number may be smaller or larger.

The results of a DNA profiling with the current technology will never give results that claim that a sample could only have come from one particular person.

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What do the DNA profiling results mean for a case?

DNA profiling does not claim to be absolute identification, but may be very strong evidence, and generally forms just one part of a case. It is really a question of looking at all the evidence in the case such as; who had the opportunity to commit the crime, eye-witness descriptions, fingerprints, the transfer of glass fragments, paint flakes or fibres linking a person to a crime and the DNA profiling results. DNA profiling is presented to the court as in the example above and the jury or magistrate can draw their own conclusions, as they do about all the evidence.

DNA profiling can be a very powerful investigative tool. Of the cases carried out so far, approximately fifty percent of the profiling results have established that the suspect was not the source of the sample associated with the crime - i.e. he/she was excluded as being the perpetrator of the crime.

Cost

It costs approximately $300.00 per sample to conduct DNA profiling. This cost covers consumables (test tubes, chemicals, etc.), overheads (electricity, heating, etc.) and the scientist's salary.

Time and procedure

It takes approximately two weeks to obtain a full DNA profile from a sample such as blood or semen. Usually many samples are analysed at one time.

However, prior to DNA profiling commencing, the case must first be processed. It must be submitted, prioritised and the individual articles must be examined by a scientist. This examination may include, depending on the circumstances of the case, analysis of hairs and fibres, identification of body fluids and analysis of blood stain patterns. DNA profiling may be just one component of the case.

When the laboratory work is completed a 'Statement' suitable for presentation in Courts of Law is written. All the work on the case then undergoes a rigorous checking procedure before it is authorised as being completed. All of these processes add to the time involved in obtaining a profile from an individual sample. There is a lot more involved than just the extraction and profiling of the DNA.

Other factors such as casework backlog, staff levels, etc. must also be taken into consideration.

Admissibility in Court

It is important to remember that in many cases DNA is not the only evidence, but some of the consequences of the DNA evidence range from charges against suspects being withdrawn, to defendants pleading guilty.

There have been a number of challenges to the DNA evidence in Victorian cases. In the first case, the DNA results were ruled inadmissible by the judge and therefore were never presented to the jury. This was not because of a problem with the scientific validity of the test, but the statistics of the result were complicated because there was not sample available from the deceased (the body was too badly decomposed) and so the scene sample was compared with samples from the parents of the deceased. The result of the trial was a verdict of guilty of homicide due to the other evidence that included a fingerprint of the accused found with the body. In this case DNA profiling was carried out using the RFLP method.

There have been no successful major challenges to DNA as being a valid technique. The reliability of the results is maintained by the stringent quality management program, which includes proficiency testing, validation studies and quality control procedures.

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Checks and balances for civil liberties

A person may voluntarily provide a sample for testing, or the Magistrates Court may issue an order directing that a sample be taken under the Crimes (Amendment) Act, 1997. This would only be done if stringent conditions are met, some of which are that;

1.    there are reasonable grounds to believe that the person has committed the offence,
2.    material reasonably believed to be from the offender is found,
3.    the taking of the sample would tend to confirm or disprove his or her involvement in the crime.

Samples are destroyed, together with any information identifying the person;

(a)     if the person is charged but not convicted - within 1 month after the conclusion of the proceedings and the end of any appeal period, or
(b)     if the person is not charged - within 12 months, unless application is made to a magistrates or Children's Court for an extension.

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Biological implications of DNA profiling

The process of DNA profiling has no biological implications or effect on the genetic make-up of either the population or the individual. It is a biological tool which reveals the genetic profile of an individual to determine whether or not he/she may be the source of biological material from a crime scene i.e. it may exclude or include an individual.

DNA profiling is also commonly in use for paternity testing, usually to determine fatherhood of a child when this is disputed.

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Some Statistics (Victoria)

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Glossary

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Links

• Gene Almanac Resources

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