Camilo Mancera A., Tarragona 20041 FORENSIC DNA Camilo Mancera Arias Ph. D. Student Rovira i Virgili University - Tarragona 2004

Camilo Mancera A., Tarragona 2004

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FORENSIC FORENSIC DNADNA

FORENSIC FORENSIC DNADNA

Camilo Mancera Arias

Ph. D. Student

Rovira i Virgili University - Tarragona 2004


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Discussion topicsDiscussion topics

• Molecular Markers

• DNA Analysis

• Paternity Test

• Genomic Pictures

• Study cases

• Final considerations


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Overview…Overview…

• In 1892, fingerprints began to be used in personal identification. The discovery that an individual's fingerprint is a unique characteristic stimulated their use for identification purposes.

• The judicial system has been able to convict a great number of suspects from fingerprints left on surfaces such as furniture, telephones, and glass. On the other hand, a comparable number of individuals have also been cleared of criminal charges because of this identification tool.

• Molecular markers can be used to characterize an individual's DNA in a pattern or profile of fragments that is unique to him or her.

• DNA analysis is transforming biological evidence as an irrefutable instrument for incrimination or absolution of suspects. It eliminates some of the ambiguities of the justice system.


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Molecular MarkersMolecular Markers

Definition: DNA Fragments that can be used as a fingerprint in the identification or characterization of individuals. There is natural genetic variation in individuals, and many genetic sequences are polymorphic, meaning they differ among individuals. Molecular markers seek to exploit this variation to identify individuals, traits, or genes on the basis of genetic differences.

Techniques

• RFLP, Restriction fragments length polymorphism.

• STR, Short tandem repeats or Microsatellites

• RADP, Random amplified polymorphism

• SNP or point mutation

• AFLP, Amplified fragment length polymorphism

• SCAR, Sequence characterized amplified regions.

PCR based techniques.


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RFLPRFLPRestriction Fragment Length PolymorphismRestriction Fragment Length Polymorphism

• Developed by Alec Jeffreys in England, in the beginning of the 1980s. • Based on the distance between restriction sites in the DNA. • RFLP uses restriction enzymes to cut DNA in specific fragments, the number and size of this fragments is unique to each individual.

Figure of Autoradiograph of a DNA cut with the enzyme Hind III and revealed by a radioactive probe (RFLP).

The relative position of bands, like a bar code, reveals the fragment sizes. The pattern of bands can then be used reliably to identify the individual source of the DNA.


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STRSTRShort Tandem RepeatsShort Tandem Repeats

• In the chromosomes of several organisms are areas containing short repeated sequence of DNA called STR, short sequence repeats (SSR), or microsatellites. • These markers group in certain areas of the genome, in blocks of tandem (one behind the other) repetitions of at least five units. • These repetitive blocks are highly variable and polymorphic, with the presence of several alleles due to the number of different repeated units.

Chromosome sequence variability at the microsatellite level

Variability in microsatellite markers for a wheat disease-resistance gene.


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RAPDRAPDRandom Amplified PolymorphismRandom Amplified Polymorphism

• It uses a synthetic oligonucleotide as a primer in the amplification process to produce a polymorphism detected by the presence or absence of a band. • This technique consists of extracting the DNA of the individuals to be analyzed and using this DNA in the amplification reactions (PCR), which can be done using a different primer for each reaction. • The products of each reaction are a result of the amplification of different chromosomal areas flanked by the pair of primers. • The amplified fragments are separated by electrophoresis in a gel.

Gel with RAPD patterns from different individuals.

Is simpler and quicker than RFLP. It uses smaller amounts of DNA, does not involve the use of radioactive probes, and is less labor intensive. It has been used mainly in plants because it does not detect variation in humans and other animals.


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DNA FingerprintDNA Fingerprint

Applications

DNA fingerprinting is also known as a DNA profile. This technique is useful in many areas, like paternity tests, criminal cases, evolution studies, evaluation of biodiversity, mapping of genes, diagnosis of inherited disorders, genetic tests, etc.

The same methods used in forensic DNA testing are also used in patent litigation to determine property rights for some crop varieties.


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DNA AnalysisDNA Analysis

• The DNA digestion with restriction enzymes produces fragments of different lengths, according to the individual's genome sequence.• The size of each fragment depends on the distance between cuts, and the number of fragments depends on the number of cut sites. (polymorphism)

Obtaining a DNA Profile (protocol)

1. Harvesting the biological sample: Blood, saliva, semen, hair, tooth, bones, or any other cellular tissue or fluid from the individual.

2. DNA extraction.3. DNA Digestion.4. Fragment Separation.5. DNA Transfer.6. Hybridization with probes.7. DNA Profile.


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DNA schematic protocolDNA schematic protocol


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Mithocondrial DNAMithocondrial DNA

When to use• In cases where the amount of DNA is extremely small • In cases where the victim has been carbonized, partially destroying the nuclear DNA.

Problem• Mitochondrial DNA tends to be much less variable, because the mitochondria is maternally inherited; that is, an individual's mitochondrial DNA is exactly identical to that of his or her mother and siblings, and to all uncles from his or her mother's side of the family, and so forth.

Study Cases• The movement known as "The Grandparents of May" in Argentina was able to reunite many children with their blood families on the basis of mitochondrial DNA analysis. • It was also used in the identification of some bodies from the World Trade Center terrorist attack on September 11, 2001 in New York City.


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Paternity TestPaternity Test

• DNA tests are the most accurate and reliable technology used for paternity identification today. Usually, samples from mother, child, and alleged father are tested to determine if the alleged father is the biological father of the child.• Paternity tests can prove with complete certainty that an individual is not a child's biological parent. However, there is no available test that can prove with 100 percent certainty that an individual is the child's biological parent. Paternity tests can guarantee at most 99 percent probability of paternity.


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Genomic PicturesGenomic Pictures

Considering that all phenotypic characteristics, such as color of eyes, hair, skin, face format, and so on, are defined by the genome, many forensic scientists believe that in the future it will be possible to substitute for the composite picture drawn based on information reported by an eyewitness with a composite drawn on the basis of the analysis of biological samples left as evidence at the scene of a crime.

Genomic picture: Reality or dream of forensic science?


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Future PossibilitiesFuture Possibilities

Some scientists believe that in the future the DNA profile will be part of the personal identification used in identity cards.


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•The first significant case of the use of DNA as criminal evidence occurred in 1986 in England, when a homicide suspect was released after DNA analysis of evidence collected at the crime scene was compared with the suspect's DNA fingerprint.

Sam Sheppard case• In December 1954, Sam Sheppard's trial in Cleveland, Ohio, occupied the media across the United States.• Convicted to life in prison for the murder of his pregnant wife, Sheppard repeatedly maintained his innocence.• In 1966, the Supreme Court threw out the trial because of trial errors. Sheppard was freed, but he died four years later.• In 1997, Sheppard's son requested authorization to exhume his father's body with the objective of obtaining a DNA analysis to substantiate his father's claims of innocence. The analysis indicated that his father's DNA did not correspond to the evidence collected at the crime scene, refuting the possibility that Sheppard had committed the crime.

Study CasesStudy Cases


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Study Cases 2Study Cases 2

Josiah Sutton case• Josiah Sutton receives a hug from his mother after he is released from a Houston jail in 2003. • Sutton served 4½ years of a 25-year prison sentence, but was exonerated after DNA evidence was introduced on appeal.


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Study Cases 3Study Cases 3

•The DNA profile of hybrid corn varieties was used in a long and controversial lawsuit between two seed companies in the United States that argued over the improper acquisition of parental inbred lines. • The DNA analysis of the inbred lines was used to settle the case. Today, many seed companies use molecular identification for its elite germplasm.


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Final ConsiderationsFinal Considerations

• Frequencies are not absolutely accurate because databases are limited

• Contamination at crime scene or from victim

• Sloppy lab management

• Chain of custody.

• Contaminated lab reagents or mislabeling of flasks.

• Civil rights issues– Establish degree of probable cause before testing DNA?– Can blood collected be used for other reasons?

Documents

Camilo Mancera A., Tarragona 20041 FORENSIC DNA Camilo Mancera Arias Ph. D. Student Rovira i Virgili University - Tarragona 2004