DNA FINGERPRINTING

This IQP was undertaken for the purpose of using the topic of DNA fingerprinting as an example of investigating the effects of technology on society. Methods for the proper collection and handling of DNA evidence were discussed, as were procedural information for the different types of DNA analysis tests. The prolonged struggle to allow DNA and other complex technical evidence in courts was highlighted through an investigation of several landmark court cases. The capabilities of DNA fingerprinting as a means of solving crimes, even those long since considered cold cases, were highlighted by reviewing a small selection of sensational court cases that involved DNA. The purposes of forensic and medical DNA databases were explained, including a discussion of the issues related to privacy rights. The report culminates in a conclusion by the authors on their findings and beliefs regarding this paramount, yet controversial, technology.

DNA, or deoxyribonucleic acid, is a complex molecule that contains all the information necessary to build and maintain an organism. It is the hereditary material. Every cell in the human body has the same DNA. The information of DNA is stored as a code constituted by four nitrogenous bases: Adenine (A), Thymine (T), Cytosine (C) and Guanine (G). The order or sequence of these bases determines the information available for building and maintaining an organism 1 . The human genome size is about 3,107 megabases (Mb) but only about 1.2 percent of the total genome encodes for proteins, this is around 20,000 genes, while 98.8 percent is noncoding DNA 2,3 , which means that do not encode proteins. Within this group we have, for example, a variable number of tandem repeats (VNTR), which are repeated sequences of 9 to 100 base pairs (bp), that play a key role in the elaboration of DNA fingerprinting. Knowing the main DNA characteristics, specificity is the key to the emergence of DNA analysis. Numerous other techniques used to determine biological markers, such as HLA and blood group substances, have been successfully applied for identification purposes. All are based on exclusion, where markers are tested until a difference is found. Other factors favoring DNA analysis include the small sample requirement, the ability to rapidly replicate a sequence a millionfold or more in vitro, and the relative stability of DNA. The point is that DNA analysis alone can be a definitive test. Once the technique becomes routine, there is little doubt that, provided a suitable specimen can be obtained, DNA fingerprinting will be the single best test for excluding a falsely associated individual 4 .

A brief history of DNA fingerprinting
In 1980, Wyman and White laid the foundations for the concept based on the observation of a polymorphic DNA locus characterized by a number of variable-length restriction fragments called restriction fragment length polymorphisms (RFLPs), which are specific sequences where restriction enzymes cleave the DNA. However, the history of DNA dates back to 1985 with the paper "Hypervariable Minisatellite Regions In Human DNA" written by Alec Jeffreys. Jeffreys and his coworkers were analyzing the human myoglobin gene when they discovered a region consisting of a 33-base-pair sequence repeated four times. This tandem repeat was referred to as a minisatellite and similar regions as hypervariable because the number of tandem repeats is variable both within a locus and between loci. In 1987, Nakamura coined the term variable number of tandem repeats (VNTR) to describe individual loci where alleles are composed of tandem repeats that vary in the number of core units. When DNA is isolated, cleaved with a specific enzyme, and hybridized under low-stringency conditions with a probe consisting of the core repeat, a complex ladder of DNA fragments is detected. This profile appears to be unique to each individual. Different core repeats were later isolated and used to produce a number of different probes useful for fingerprinting 4 . For that time, this technique was unknown but its potential was evident. DNA fingerprinting had its first application in 1985 in a case of parentage testing, actually a maternity test, with paternal DNA unavailable. In this unusual case, a mother with her little 13 years old son were arrested in the airport when they arrived in England from Ghana because the authorities thought that he was not her son. A DNA fingerprinting applied to both demonstrated that, effectively, they told the truth. The first application of DNA fingerprinting in forensic identification happened later that same year, in a case that beautifully exemplifies the power of DNA evidence to link crime-scenes, to exclude suspects, and to support convictions. A suspect was arrested for allegedly committing a double rape and suicide to 2 minors 5 . A DNA fingerprinting using a sample of semen left in the crime scene demonstrated that a man had been responsible for both crimes but it was not the arrested suspect. He was released and the real culprit was arrested. Nowadays, this technique is still used to create DNA profile of each individual in order to clarify some crimes or parentage testing 6 .

What is DNA fingerprinting?
In simple words, DNA Fingerprinting is the technology which is used to identify individuals on the basis of the molecular characteristics of the DNA7. More specific, this method uses VNRT because the number of bases and repeats within a locus is unique to each individual. For example, an individual can have in his genome the sequence gatagata and this repeats 10 times and another can have the same sequence but only repeats 5 times. The technique is used, as we have seen before, in parentage testing and forensic cases but it can be used for anthropological genetics, zoology, and botany among others disciplines. Importantly, the technique of DNA Fingerprinting is very sensitive, which means that it can also generate data even from half (partially) decomposed biological material 7 .
Procedure to create a DNA fingerprinting.
The steps involve others techniques used in Molecular Biology, such as polymerase chain reaction (PCR) and electrophoresis among others. The following are the steps to generate a DNA fingerprinting.
1. The DNA is extracted from the nuclei of any cell in the body. 2. The DNA molecules are broken with the help of enzyme restriction endonuclease (called chemical knife) that cuts them into fragments. The fragments of DNA also contain the VNTRs. 3.
The fragments are separated according to size by gel electrophoresis in agarose gel. 4.
The separated fragments of single-stranded DNA are transferred onto a nylon membrane. Radioactive DNA probes having repeated base sequences complementary to possible VNTRs are poured over the nylon membrane. Some of them will bind to the of single-stranded VNTRs. The method of hybridization of DNA with probes is called Southern Blotting. 5. The nylon membrane is washed to remove extra probes.
6. An X-ray film is exposed to the nylon membrane to mark the places where the radioactive DNA probes have bound to the DNA fragments. These places are marked as dark bands when X-ray film is developed. This is known as autoradiography. 7. The dark bands on X-ray film represent the DNA fingerprints (DNA profiles) 5 .
These steps are shown better in figure 1.

DNA Fingerprinting applications
Since Alec Jeffreys developed the DNA fingerprinting technique, it has been used in different scientific fields. In forensic investigations has helped to send to prison criminals, and identify victims of crimes, natural disaster, wars. Paternity disputes have been resolved thanks to this method. Moreover, disciplines as anthropological genetics, zoology, and botany among others have driven profiling research in order to interpret the origin and behavior of some species. In the next lines, we are going to describes how the technique has been applied and evolved in the areas mentioned above.

Forensic Investigations
Famous Crime T.V shows as CSI, Bones and others have popularized this technology. To summarize the methodology, genetic material like blood, semen, saliva, hair and skin found at the crime scene are processed, and afterward the samples are compared with the DNA of the suspects, in order to determine guilt or innocence of the accused. DNA fingerprinting markers have evolved since 1984. In the beginning, sets of minisatellites or oligonucleotides stretches were used, also called multi-locus probes (MLP) which detected sets of 15 to 20 variable fragments per individual ranging from 3.5 to 20 kb in size. Minisatellites were replaced because they needed a large amount of molecular weight of DNA, usually not found at the crime scene and errors in the linkage between loci. For this reason was changed by single locus probe (SLP) which recognized single hypervariable locus, using high stringency hybridization and just 10 ng of DNA8. Multilocus and Single Locus probes were part of the socalled restriction fragment length polymorphism (RFLP)based methods were still limited by the available quality and quantity of the DNA. Those procedures were replaced by PCRbased methods because they improved sensitivity, speed, and genotyping precision. PCR-based methods use microsatellites as markers instead of minisatellites; microsatellites as short tandem repeats (STRs) are more sensitive and less prone to allelic dropout than VNTR (variable number of tandem repeat) systems8. In cases, when there exist a low proportion of nuclear DNA samples, lineage marker is used which are obtained from mitochondrial and Y DNA, and they are very useful to reconstruct the paternal and maternal relationship and historical reconstruction in unidentified remains typically skeletonized, hair shafts without roots, or very old specimens where only heavily degraded DNA is available likewise samples of sexual assault without ejaculation, sexual assault by a vasectomized male, male DNA under the fingernails of a victim, male 'touch' DNA on the skin8.

Parentage testing
DNA fingerprinting is an advantageous technique in cases, such as, of establishing the paternity of disputed offspring or cases of baby swapping. This method replaced ABO blood antigen systems which cannot establish paternity but can conclusively exclude an alleged father from being a candidate. Disputed paternity originates because of affiliation orders, divorce proceedings and questioned the legitimacy, also is used to discover paternity in cases of inheritance, guardianship, maintenance, legitimacy, adultery or fornication9. In Parentage testing, a DNA comparison is performed between progeny against potential parents. Children inherit half of their alleles from each parent and thus should possess an alleles combination of their parents. Anthropological genetics In anthropological genetics, markers have been used as ancestryinformative markers to reconstruct the human diaspora and to interpret the evolutionary history of human populations to inquire population origins, migration, admixture and adaptation to different environments, as well as susceptibility and resistance to disease 10 .
In the medical field, researchers have made possible the mapping quantitative trait loci involved in biological pathways of diseases such as diabetes mellitus, cancers, obesity, osteoporosis, and coronary heart disease. In the studies of population, markers allow identifying the presence, absence, or high frequency in some populations and low frequencies in others, of certain genetic traits that characterize some specific population 11 .

Botany
DNA fingerprinting is an essential tool for genotype identification in both wild plant and cultivated species. DNA profiling is used for protection of biodiversity, identifying markers for traits, identification of gene diversity and variation 12  DNA markers help to study fundamental evolutionary influences of natural selection, mutation, gene flow and genetic drift on wild plant populations and identify groups are characterized by highly variable ploidy levels, often even within the same species. Moreover, the method detects both ancient and ongoing hybridization between crops and wild species 12 .

Zoology
In Zoology, DNA fingerprinting determine the genetic identity of individuals and measure genetic variation in natural populations, allowing true genetic relationships among individuals to be determined, rather than them being inferred from field observations. Furthermore, it helps to test predictions of kin selection models in a realistically way, and detect hybrids species 13 .
The DNA marker clarified mating system in reproductive ecology for example in vertebrates that give birth to more than one offspring has revealed concurrent multiple paternities. This kind of behavior has been observed in a wide range of organisms, particularly in reptiles.