How does forensic identification work?

Any type of organism can be identified by examination of DNA sequences unique to that species. To identify individuals, forensic scientists scan 13 DNA regions that vary from person to person and use the data to create a DNA profile of that

individual (sometimes called a DNA fingerprint). There is an extremely small chance that another person has the same DNA profile for a particular set of

regions.

Some Examples of DNA Uses for Forensic Identification

Identify potential suspects whose DNA may match evidence left at crime scenes Exonerate persons wrongly accused of crimes

Identify crime and catastrophe victims Establish paternity and other family relationships

Match organ donors with recipients in transplant programs

DNA FORENSICS

Is DNA effective in identifying persons?

Consider the scenario of a crime scene investigation Assume that type O blood is found at the crime scene. Type O occurs in about

45% of Americans. If investigators type only for ABO, then finding that the "suspect" in a crime is type O really doesn't reveal very much.

If, in addition to being type O, the suspect is a blond, and blond hair is found at the crime scene, then you now have two bits of evidence to suggest who really

did it. However, there are a lot of Type O blonds out there.

If you find that the crime scene has footprints from a pair of Nike Air Jordans (with a distinctive tread design) and the suspect, in addition to being type O and blond, is also wearing Air Jordans with the same tread design, then you are much closer

to linking the suspect with the crime scene.

In this way, by accumulating bits of linking evidence in a chain, where each bit by itself isn't very strong but the set of all of them together is very strong, you can

argue that your suspect really is the right person.

How is DNA typing done?

•About 3 million bases differs from one person to the next. •Scientists can use these variable regions to generate a DNA profile of an

individual, using samples from blood, bone, hair, and other body tissues and products.

In criminal cases, this generally involves obtaining samples from crime-scene evidence and a suspect, extracting the DNA, and analyzing it for the presence of a

set of specific DNA regions (markers).

•Scientists find the markers in a DNA sample by designing small pieces of DNA (probes) that will each seek out and bind to a complementary DNA sequence in

the sample. A series of probes bound to a DNA sample creates a distinctive pattern for an individual.

•Forensic scientists compare these DNA profiles to determine whether the suspect's sample matches the evidence sample. A marker by itself usually is not

unique to an individual; if, however, two DNA samples are alike at four or five regions, odds are great that the samples are from the same person.

•If the sample profiles don't match, the person did not contribute the DNA at the crime scene.

•The more probes used in DNA analysis, the greater the odds for a unique pattern and against a coincidental match, but each additional probe adds greatly to the time and expense of testing. •A minimum of six probes are recommended. DNA chip technology (in which thousands of short DNA sequences are embedded in a tiny chip) will enable much more rapid analysis using many more probes, and

raising the odds against coincidental matches.

Some of the DNA technologies used in forensic investigations

Restriction Fragment Length Polymorphism (RFLP)RFLP is a technique for analyzing the variable lengths of DNA fragments that result from

digesting a DNA sample with a special kind of enzyme. This enzyme, a restriction endonuclease, cuts DNA at a specific sequence pattern know as a restriction

endonuclease recognition site. The presence or absence of certain recognition sites in a DNA sample generates variable lengths of DNA fragments, which are separated using

gel electrophoresis. They are then hybridized with DNA probes that bind to a complementary DNA sequence in the sample.

RFLP is one of the original applications of DNA analysis to forensic investigation. With the development of newer, more efficient DNA-analysis techniques, RFLP is not used as

much as it once was because it requires relatively large amounts of DNA. In addition, samples degraded by environmental factors, such as dirt or mold, do not work well with

RFLP.

PCR AnalysisPCR (polymerase chain reaction) is used to make millions of exact copies of DNA from a biological sample. DNA amplification with PCR allows DNA analysis on biological samples as small as a few skin cells. The ability of PCR to amplify such tiny quantities of DNA enables even highly degraded samples to be analyzed. Great care, however, must be taken to prevent contamination with other biological materials during the identifying, collecting, and preserving of a sample.

STR Analysis

Short tandem repeat (STR) technology is used to evaluate specific regions (loci) within nuclear DNA. Variability in STR regions can be used to distinguish one DNA profile from another. The Federal Bureau of Investigation (FBI) uses a standard set of 13 specific STR regions for CODIS. CODIS is a software program that operates local, state, and national databases of DNA profiles from convicted offenders, unsolved crime scene evidence, and missing persons. The odds that two individuals will have the same 13-loci DNA profile is about one in one billion. STRs are currently the most popular type of DNA fingerprint, since the wholePCR process takes only a few hours, compared to RFLP/VNTR probe hybridization and film exposure which can take several days. STRs can use much smaller samples of DNA than RFLPs/VNTRs, and can even use partially degraded DNA to create a fingerprint.

Thus, the integrity and quality of the DNA sample is not as great a factor with STRs than with the traditional methods of DNA fingerprinting. The current standard forensic protocol analyses 13 core STR loci which have been carefully chosen for their uniqueness. The only disadvantage of the STR approach is it is sensitive to contaminating DNA, so usually the STR approach is used first, followed by a VNTR analysis if contamination is suspected, and enough DNA is available.

Mitochondrial DNA Analysis

Mitochondrial DNA analysis (mtDNA) can be used to examine the DNA from samples that cannot be analyzed by RFLP or STR. Nuclear DNA must be extracted from samples for use in RFLP, PCR, and STR.

While older biological samples that lack nucleated cellular material, such as hair, bones, and teeth, cannot be analyzed with STR and RFLP, they can be analyzed with mtDNA. In the investigation of cases that have gone unsolved for many years, mtDNA is extremely valuable.

All mothers have the same mitochondrial DNA as their daughters. This is because the mitochondria of each new embryo comes from the mother's egg cell. The father's sperm contributes only nuclear DNA.

Comparing the mtDNA profile of unidentified remains with the profile of a potential maternal relative can be an important technique in missing person investigations.

Y-Chromosome Analysis

The Y chromosome is passed directly from father to son, so the analysis of genetic markers on the Y chromosome is especially useful for tracing relationships among males or for analyzing biological evidence involving multiple male contributors.

VNTRs - σημαντικοί γενετικοί μάρτυρες για εγκληματολογικές αναλύσεις

•Το μέγεθος του ενισχυμένου τμήματος ή αλληλομόρφου εξαρτάται από τον αριθμό και το μέγεθος των επαναλαμβανόμενων αλληλουχιών που περιέχονται σε αυτό.

•Με κατάλληλους VNTR γενετικούς τόπους και υψηλής διακριτικής ικανότητας ηλεκτροφορητικά συστήματα, η ενίσχυση συγκεκριμένων VNTR αλληλουχιών με PCR μπορεί να φανεί χρήσιμη για αναλύσεις ταυτοποίησης.

Legend:

(A)The DNA sequences that create the variability used in this analysis contain runs of short, repeated sequences, such as GTGTGT . . . , which are found in various positions (loci) in the human genome. The number of repeats in each

run is highly variable in the population, ranging from 4 to 40 in different individuals. A run of repeated nucleotides of this type is commonly referred to

as a VNTR (variable number of tandem repeat) sequence. Because of the variability in these sequences, each individual will usually inherit a different

variant of each VNTR locus from their mother and from their father; two unrelated individuals will therefore not usually contain the same pair of

sequences. A PCR reaction using primers that bracket the locus produces a pair of bands of

amplified DNA from each individual, one band representing the maternal variant and the other representing the paternal variant. The length of the

amplified DNA, and thus its position after electrophoresis, will depend on the exact number of repeats at the locus.

(B) The DNA bands obtained from a set of four different PCR reactions, each of which amplifies the DNA from a different VNTR locus. In the schematic example shown here, the same three VNTR loci are analyzed from three

suspects (individuals A, B, and C), giving six bands for each person. You can see that although some individuals have several bands in common, the overall

pattern is quite distinctive for each.

The band pattern can serve as a “fingerprint” to identify an individual nearly uniquely. The fourth lane (F) contains the products of the same PCR reactions carried out on a forensic sample. The starting material for such a PCR reaction can be a single hair that was left at the scene of the crime. From the example, individuals A and C can be eliminated from enquiries, while B remains a clear suspect.

One of the first accepted uses of DNA fingerprinting was in the investigation ofsexual assault and rape cases. Detectives only had to match the DNA of the semen foundat the scene of the crime with the DNA of any potential suspect to determine who wasguilty of committed the crime. A DNA sample from the rapist could be obtained from asimple vaginal swab from the victim or any other semen that was released in the areaduring the assault. The figure below shows how a DNA fingerprint can help determinewho is guilty of a sexual assault.

As seen from figure below , suspect B (lane 4) is guilty of rape because his DNA fragmentsmatch that of the semen found on the victim’s clothes (lane 3) and also in the vagina(lane 6). Suspect A (lane 2) is clearly not the rapist because his DNA fragments do notmatch the semen found on the victim’s clothes or the semen from the vaginal swab.DNA fingerprinting is very useful in such an application because it provides the policewith an exact match of who left evidence at the crimescene.

In paternity tests potential fathers of the child have theirDNA analyzed with the child and mother’s DNA in order to see which of the potential fathers has the most DNA in common with the child in question. Figure 6 shows an example of a RFLP used to determine which potential father (F1 and F2) is the real father of the child (C). As you can see in the figure below, the second father tested (F2) seems to have more DNA in common with the child than that of the first father tested (F1).

Figure 6. A paternity test usingthe RFLP technique

DNA can be obtained from traces of biological fluids or tissues at a crime scene.The most traditional sources of forensic DNA come from saliva, seminal fluid, blood, and hair. DNA from saliva can be extracted from items such as cigarette butts, ski masks, envelopes, and stamps. Seminal fluids are usually found from oral, rectal, or vaginal swabs, and also on clothing. Blood stains and hair that are visible at a crime scene usually contain DNA that can be analyzed at the forensic laboratory

Contamination is one of the greatest risks that the evidence must be guarded from.If your sample of evidence is found to be contaminated, it can be thrown out as evidence in the courtroom. Contamination can occur at the crime scene, during packaging, in transit to the laboratory, and also during analysis. With a risk of possible contamination present in all these steps of the forensic process, proper precautions must be used to prevent ruining the DNA sample.At the crime scene many factors must be considered in trying to preventcontamination. The first factor is Nature. For example, if it rained at the crime scene,a blood stain found could be diluted which would be almost impossible to analyze. Also if it was windy that day then vital pieces of DNA could have been blown away from the crime scene. Another factor at the crime scene is properly securing the area so that people do not taint the evidence. Until a crime scene is secured many individuals not related to the event may have left DNA around key evidence which maybe mistaken for a possible suspect. Equipment is another factor which must be regulated to reduce the risk of evidence contamination.

Clothing, notepads, photography equipment, and crime scene kits must be properly decontaminated once leaving a crime scene or they may contaminate evidence at another crime scene. Disposable personal protective equipment (PPE) should be worn including: a mask, jumpsuit, gloves, booties and head cover. By keeping these tips in mind, contamination at a crime scene should be at a minimum.

During packaging and transport of the DNA evidence, there are also a few factorsto keep in mind in order to preserve the sample in its original state. When packaging evidence, each sample should be sealed individually to prevent cross-contamination.Biological fluids should be dried in order to avoid contamination by bacteria . Evidence should be packaged in a paper container, which is then put into an openplastic container to reduce the risk of evidence leaking out while in transit. When the biological sample is in transit certain precautions must be taken. DNA is sensitive to temperature which means that while the sample is in transit, the package must be out of direct sunlight or any other extreme circumstance.

The samples acquired from the crime scene should be kept away from high temperatures. Room temperature is acceptable, refrigeration is desirable, and freezing is preferred when 20 it comes to storing a sample.

DNA at an Aged Crime SceneAt an aged crime scene, forensic scientists attempt to retrieve DNA evidence thatis the least contaminated and decomposed. For example, if a deceased individual is at an aged crime scene and is showing signs of decomposition, then blood work would not be a practical route of obtaining a valid DNA sample .If hair is present on the deceased body, the sample must be pulled from the tissue because the tissue attached to the hair root is necessary for DNA analysis. Bones and teeth would also serve as a suitable sample of DNA evidence, since they take very long to degrade.

We now know that tiny particles of biological fluids, most notably blood, can cling to most surfaces at the crime scene for many years without anyone ever realizing they are there . There is now a chemical that allows you to see these microscopic particles of blood and find the clues, that to the naked eye,did not seem to exist. The chemical is called luminol and it reveals these particles with alight-producing chemical reaction between hemoglobin and several chemicals. Light is produced because the reactants contain more energy than the products of the reaction, which release the extra energy in the form of light photons. This is the process, which is called chemiluminescence. After the chemicalis sprayed onto the object in question, a blue glow will emit in any areas where there aretiny traces of blood, an example of which is shown in figure 4.

A simulation of luminol at work. The chemical binds to the blood and emits a blue glow. Once the luminol reacts with the small traces of blood left behind, forensic investigators will takes pictures or videotape the crime scene to record any potential patterns . It should be noted that once blood reacts with luminol, that sample is no longer able to provide DNA for analysis. Perhaps in the future a new method of visualization will be devised that maintains DNA structure.

CONCLUSIONS

DNA fingerprinting is the most sophisticated way to identify living organisms.DNA cannot easily be altered once it is left at a crimescene or deposited with a mummy, which makes it a strong forensic tool. RFLPs and VNTRs are the traditional methods of fingerprinting DNA, which uses a relatively large sample that uses the method of probe hybridization to detect polymorphisms in the DNA. STRs are the most current form of DNA fingerprinting, which is PCR based anduses a very small sample of DNA. DNA fingerprinting has many applications that range from criminal rape cases, paternity tests, molecular archeology etc.DNA forensics is one of the greatest tools in piecing together a crime scene. Overthe past ten years there have been many advances in the methods of collecting and preserving these DNA samples to help facilitate the acceptance of this evidence in the court room. By avoiding contamination and properly storing it to prevent degradation,forensic science has made a monumental step in allowing DNA samples as valid evidence in United States courtrooms. DNA evidence is now one of the most powerful tools used in determining who is responsible for a crime. With criminals altering their fingerprints and other physical characteristics, DNA evidence is one of the only true methods to correctly identify an individual. Now with the help of chemicals such as luminol, crime scenes that at first analysis seem to have no physical evidence are further examined on the particle level which makes it almost impossible to leave a crime without a trace.

DNA Forensics Databases National DNA Databank: CODIS

The COmbined DNA Index System, CODIS, blends computer and DNA technologies into a tool for fighting violent crime. The current version of CODIS uses two indexes to generate investigative leads in crimes where biological evidence is recovered from the crime scene. The Forensic index contains DNA profiles developed from crime scene evidence. All DNA profiles stored in CODIS are generated using STR (short tandem repeat) analysis.

CODIS utilizes computer software to automatically search its two indexes for matching DNA profiles. Law enforcement agencies at federal, state, and local levels take DNA from biological evidence (e.g., blood and saliva) gathered in crimes that have no suspect and compare it to the DNA in the profiles stored in the CODIS systems. If a match is made between a sample and a stored profile, CODIS can identify the perpetrator.

All 50 states have laws requiring that DNA profiles of certain offenders be sent to CODIS. As of January 2003, the database contained more than a million DNA profiles in its Convicted Offender Index and about 48,000 DNA profiles collected from crime scenes but which have not been connected to a particular offender.

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In March 2003 USA goverment proposed $1 billion in funding over 5 years to reduce the DNA testing backlog, build crime lab capacity, stimulate research and

development, support training, protect the innocent, and identify missing persons.

More on CODIS CODIS: Combined DNA Index System - Information from the FBI.

The FBI Laboratory's Combined DNA Index System Program - Enter regional information to learn more about CODIS in your area of the world. From Promega Corporation, a major supplier of reagents and other

materials to support molecular biology research.

National Commission on the Future of DNA Evidence

Postconviction DNA Testing: Recommendations for Handling Requests - Report from the National Commission on the Future of DNA Evidence

Another application of DNA fingerprinting is a more recent method in moleculararcheology. This method of archeology uses DNA to determine a species of anarcheological discovery or to trace blood lines of animal or human remains. DNA may be extracted from biological remains, hair, teeth, body tissues, or even fossils. The best climates to preserve DNA are very cold temperatures and arid climates. Some examples of specimens from these types of climates are the “Tyrolean Ice-Man”, who was found in the Alps, and the mummies of Egypt found in the dry desert.The ice man was found to be around 5300 years old, and DNA was extracted from the remains of his gut which found small traces of food that he ate (Ice Man, 2005). This was one of the most historic archeological discoveries in the last century. DNA fingerprinting is an important tool for archeologists to piece together information that links the past to us today.