Variables o indicadores

In 1985 a publication in the journal Nature denoted that the analysis of several tandem repeats on the DNA sequence of humans “…are a powerful method for paternity and maternity testing, can be used in forensic applications and might also be useful in detecting inbreeding…”. In the same article, Dr. Alec Jeffreys called these sequences “DNA fingerprints” (59). Following this discovery, ‘DNA fingerprinting’ was first used in a murder case the U.K. by Sir Alec Jeffreys to help determine who committed the rape and murder of two young girls. DNA was able to connect both crimes showing that the body fluids found at the crime scene were from the same perpetrator. Dr. Jeffreys was the pioneer of ‘DNA fingerprinting’ and its use spread to Europe and all over the world (60). The DNA fragments, called fingerprints, are also known as RFLP (restriction fragment length polymorphisms) and were obtained by incubation of DNA with restriction enzymes that specifically cut the DNA in the same consensus sequences throughout the DNA strand of interest. In the USA the enzyme most commonly used was HaeIII which recognizes and cuts the sequence ‘GGCC’ (61). The RFLP method as used by Dr.

Jeffreys first extracts the DNA by lysing the cells and disrupting membranes and proteins attached to the DNA. Next, the restriction enzymes are incubated with the extracted DNA and the fragments they produce are separated by size through the use of an agarose gel. In order to determine which fragments were present, the gel product was transferred to a nylon membrane, the double stranded was then denatured by a strong alkaline solution and the bands hybridized with DNA probes. The specific base pairing between the bands

in the nylon membrane and the nucleotide sequence in the radiolabelled probes allowed the detection of specific RFLP fragments. The membrane was placed next to X-ray film and the location of the RFLP bands were photographed (62). In the original method, the probes hybridized to several DNA bands creating a unique pattern that could be

compared with a control sample. However, since forensic samples often are composed of mixtures, the bands would be the result of DNA from different contributors, which could be difficult to analyze. The technology then evolved to use single-locus probes that hybridize only to a single locus and give one band for homozygous or two bands for heterozygous individuals (60). To analyze the presence of another RFLP, the process was repeated by first removing the first single-locus probe by a alkaline solution washing, and then hybridizing the membrane with new single-locus probe and a photograph taken. The analysis of multiple RFLPs could take several weeks or even months to be completed. Besides the low throughput, this method was also limited to samples with high yields of DNA (>50 ng) and good quality and high molecular weight (>12 kb) DNA (63).

Kary Mullis had already invented the method referred to as polymerase chain reaction (PCR) in 1986 (64). However it was not immediately used in forensics. PCR has a great advantage since it mimics in vitro what occurs with DNA replication in vivo. Briefly, the extracted DNA is incubated with a DNA polymerase, primers that flank the region of interest to be copied, deoxyribonucleotides (dNTPs) and ions such as

magnesium which are necessary for enzymatic activity. The PCR product consists of multiple copies of the region of interest, which is delimited by the primer sequences.

In 1990, some laboratories were using dot blot tests which work by hibridizing the denatured PCR product with specific probes captured in a nylon membrane (65).

Colorimetric detection allowed the appearance of blue dots for positive hybridization. Even though there were issues with low power of discrimination of the method and the possibility that the PCR products would re-anneal prior to hybridization with the probes, the use of the method allowed the introduction of PCR-based technology in the court system.

Once the sequence of the regions used in RFLP were further explored, several attempts tried to perform PCR to amplify each of the RFLPs. In 1994, the D1S80 kit allowed the amplification by PCR of the locus D1S80 which spanned 400-800 bp,

depending on the number of repeats present in the individual DNA sample (66). The PCR products were analyzed by a vertical polyacrylamide gel followed by silver stain

detection. However, the relatively large length of fragments, made it still difficult to type samples with degraded DNA. The main limitations of the methods at that point were either the need to use long sequences of DNA making it problematic to analyze degraded samples and presenting a low power of discrimination. Most importantly, none of the methods studied were easily automatable or allowed a high throughput analysis.

Even though short-tandem repeats (STRs) were known since the late 1980’s, in 1995 the United Kingdom Forensic Science service launched the use of 6 STRs for suspect match in forensic analysis (60). STRs are regions in the genome that show smaller repeat units (2 to 7 bp), therefore called ‘microsatellites’ when compared to the 9- 100 bp found in the minisatellites typed with RFLP. In 1997, the FBI laboratory selected the 13 USA core STRs as the necessary loci to be typed for DNA forensic analysis and stored in a national database. Even though the small size of STRs compared to RFLP allowed the use of degraded DNA, the analysis step still relied on silver staining of

polyacrylamide gels thus not allowing high throughput. Another issue was that the same DNA sample would have to be divided into 13 separate PCR reactions for each one of the 13 loci and their analysis was done separately on in groups of two or three. To overcome such difficulty, a multiplex PCR reaction was developed allowing the amplification of several STRs. Presently the multiplex amplification and analysis of 15 STRs allow a power of discrimination of 1 in a trillion or greater (Table 1.1(67)).

Table 1.1 Summary of characteristics comparing RFLP and methods based on PCR, such as STR typing. Adapted from (67)

Characteristic RFLP Methods PCR Methods

Time required to obtain

results 6-8 weeks with radioactive _{probes; ∼ 1 week with} chemiluminiscent probes

1-2 days

Amount of DNA needed 50-500 ng 0.1-1 ng

Condition of DNA needed

High-molecular-weight, intact DNA

May be highly degraded Capable of handling

sample mixtures Yes (single-locus probes) Yes Allele identification Binning required since a

distribution of sizes are observed

Discrete alleles obtained

Form used in analysis DNA must be double stranded for restriction enzymes to work

DNA can be either single stranded or double stranded

Power of discrimination _{∼ 1 in 1 billion with 6}

loci ∼ 1 in 1 billion with 8 to 13 loci (requires more loci)

Automatable and capable of high-volume sample processing