| Genetic fingerprinting, DNA testing, DNA | | | | automated, and allows for easy creation |
| typing, and DNA profiling are techniques | | | | of phylogenetic trees based on comparing |
| used to distinguish between individuals | | | | individual samples of DNA. Due to its |
| of the same species using only samples | | | | relatively low cost and ease of set-up |
| of their DNA. Its invention by Sir Alec | | | | and operation, AmpFLP remains popular in |
| Jeffreys at the University of Leicester | | | | lower income countries. |
| was announced in 1985. Two humans will | | | | STR analysis |
| have the vast majority of their DNA | | | | The most prevalent method of DNA |
| sequence in common. Genetic | | | | fingerprinting used today is based on |
| fingerprinting exploits highly variable | | | | PCR and uses short tandem repeats (STR). |
| repeating sequences called | | | | This method uses highly polymorphic |
| minisatellites. Two unrelated humans | | | | regions that have short repeated |
| will be likely to have different numbers | | | | sequences of DNA (the most common is 4 |
| of minisatellites at a given locus. By | | | | bases repeated, but there are lengths in |
| using PCR enough DNA is obtained to then | | | | use, including 3 and 5 bases). Because |
| detect the number of repeats at several | | | | different people have different numbers |
| loci. It is possible to establish a | | | | of repeat units, these regions of DNA |
| match that is extremely unlikely to have | | | | can be used to discriminate between |
| arisen by coincidence, except in the | | | | individuals. These STR loci (locations) |
| case of identical twins, who will have | | | | are targeted with sequence-specific |
| identical genetic profiles. | | | | primers and are amplified using PCR. The |
| Genetic fingerprinting is used in | | | | DNA fragments that result are then |
| forensic science, to match suspects to | | | | separated and detected using |
| samples of blood, hair, saliva or semen. | | | | electrophoresis. There are two common |
| It has also led to several exonerations | | | | methods of separation and detection, |
| of formerly convicted suspects. It is | | | | capillary electrophoresis (CE) and gel |
| also used in such applications as | | | | electrophoresis. |
| identifying human remains, paternity | | | | The polymorphisms displayed at each STR |
| testing, matching organ donors, studying | | | | region are by themselves very common, |
| populations of wild animals, and | | | | typically each polymorphism will be |
| establishing the province or composition | | | | shared by around 5 - 20% of individuals. |
| of foods. It has also been used to | | | | When looking at multiple loci, it is the |
| generate hypotheses on the pattern of | | | | unique combinations of these |
| the human diaspora in prehistoric times. | | | | polymorphisms to an individual that |
| Testing is subject to the legal code of | | | | makes this method discriminating as an |
| the jurisdiction in which it is | | | | identification tool. The more STR |
| performed. Usually the testing is | | | | regions that are tested in an individual |
| voluntary, but it can be made compulsory | | | | the more discriminating the test |
| by such instruments as a search warrant | | | | becomes. |
| or court order. Several jurisdictions | | | | From country to country different STR |
| have also begun to assemble databases | | | | based DNA profiling systems are in use. |
| containing DNA information of convicts. | | | | In North America CODIS is prevalent, |
| The United Kingdom currently has the | | | | while in the UK the SGM+ system, which |
| most extensive DNA database in the | | | | is compatible with The National DNA |
| world, with well over 2 million records | | | | Database is in use. Whichever system is |
| as of 2005: The National DNA Database | | | | used, many of the STR regions under test |
| (NDNAD). The size of this database, and | | | | are the same. These DNA profiling |
| its rate of growth, is giving concern to | | | | systems are based around multiplex |
| civil liberties groups in the UK, where | | | | reactions, whereby many STR regions will |
| police have wide-ranging powers to take | | | | be under test at the same time. |
| samples and retain them even in the | | | | Capillary electrophoresis works by |
| event of acquittal. | | | | electrokinetically (movement through the |
| DNA identification must be done by an | | | | application of an electric field) |
| extraction of DNA from substances such | | | | injecting the DNA fragments into a thin |
| as: | | | | glass tube (the capillary) filled with |
| Personal items (e.g. toothbrush, razor, | | | | polymer. The DNA is pulled through the |
| ...) | | | | tube by the application of an electric |
| Banked samples (e.g. banked sperm or | | | | field, separating the fragments such |
| biopsy tissue) | | | | that the smaller fragments travel faster |
| Blood kin (biological relative) | | | | through the capillary. The fragments are |
| Human remains previously identified | | | | then detected using fluorescent dyes |
| Reference samples are often collected | | | | that were attached to the primers used |
| using buccal swab. | | | | in PCR. This allows multiple fragments |
| DNA fingerprinting methods | | | | to be amplified and run simultaneously, |
| DNA fingerprinting begins by extracting | | | | something known as multiplexing. Sizes |
| DNA from the cells in a sample of blood, | | | | are assigned using labeled DNA size |
| saliva, semen, or other appropriate | | | | standards that are added to each sample, |
| fluid or tissue. | | | | and the number of repeats are determined |
| RFLP analysis | | | | by comparing the size to an allelic |
| When DNA fingerprinting first began, | | | | ladder, a sample that contains all of |
| restriction fragment length polymorphism | | | | the common possible repeat sizes. |
| (RFLP) analysis was used, though it has | | | | Although this method is expensive, |
| been almost completely replaced with | | | | larger capacity machines with higher |
| newer techniques. RFLP analysis is | | | | throughput are being used to lower the |
| performed by using a restriction enzyme | | | | cost/sample and reduce backlogs that |
| to cut the DNA into fragments which are | | | | exist in many government crime |
| separated into bands during agarose gel | | | | facilities. |
| electrophoresis. Next, the bands of DNA | | | | Gel electrophoresis acts using similar |
| are transferred via a technique called | | | | principles as CE, but instead of using a |
| Southern blotting from the agarose gel | | | | capillary, a large polyacrylamide gel is |
| to a nylon membrane. This is treated | | | | used to separate the DNA fragments. An |
| with a radioactively-labeled DNA probe | | | | electric field is applied, as in CE, but |
| which binds to certain specific DNA | | | | instead of running all of the samples by |
| sequences on the membrane. The excess | | | | a detector, the smallest fragments are |
| DNA probe is then washed off. An X-ray | | | | run close to the bottom of the gel and |
| film placed next to the nylon membrane | | | | the entire gel is scanned into a |
| detects the radioactive pattern. This | | | | computer. This produces an image showing |
| film is then developed to make a visible | | | | all of the bands corresponding to |
| pattern of bands called a DNA | | | | different repeat sizes and the allelic |
| fingerprint. By using multiple probes | | | | ladder. This approach does not require |
| targeting various polymorphisms in | | | | the use of size standards, since the |
| successive X-ray images, a fairly high | | | | allelic ladder is run alongside the |
| degree of discrimination was possible. | | | | samples and serves this purpose. |
| The primary drawback of RFLP is that the | | | | Visualization can either be through the |
| exact sizes of the bands are unknown and | | | | use of fluorescently tagged dyes in the |
| comparison to a molecular weight ladder | | | | primers or by silver staining the gel |
| is done in a purely qualitative manner. | | | | prior to scanning. Although it is cost |
| Many labs developed policies that | | | | effective and can be rather high |
| described what they considered a unique | | | | throughput, silver staining kits for |
| band, but it was not standardized and | | | | STRs are being discontinued. In |
| led to DNA fingerprinting coming under | | | | addition, many labs are phasing out gels |
| harsh attack in People v. Castro 545 | | | | in favor of CE as the cost of machines |
| N.Y.S. 2d. 985 (Sup. Ct. 1989). RFLP was | | | | becomes more manageable. |
| a very time consuming method which | | | | The true power of STRs is in its |
| required relatively high quantity of | | | | statistical power of discrimination. In |
| good quality DNA to be used (such as a | | | | the U.S.A., there are 13 loci (DNA |
| dime sized blood drop). This made typing | | | | locations) that are currently used for |
| degraded samples such as those from | | | | discrimination. Because these loci are |
| evidence that had been exposed to the | | | | independently assorted (having a certain |
| elements fairly difficult. | | | | number of repeats at one locus doesn't |
| PCR analysis | | | | change the likelihood of having any |
| With the invention of polymerase chain | | | | number of repeats at any other locus), |
| reaction (PCR), DNA fingerprinting took | | | | the power rule of statistics can be |
| huge strides forward in both | | | | applied. This means that if someone has |
| discriminating power and ability to | | | | the DNA type of ABC, where the three |
| recover information from very small | | | | loci were independent, we can say that |
| starting samples. PCR involves the | | | | the probability of having that DNA type |
| amplification of specific regions of DNA | | | | is the probability of having type A |
| using a cycling of temperature and a | | | | times the probability of having type B |
| thermostable polymerase enzyme along | | | | times the probability of having type C. |
| with sequence specific primers of DNA. | | | | This has resulted in the ability to |
| Commercial kits that used single | | | | generate match probabilities of 1 in a |
| nucleotide polymorphisms (SNPs) for | | | | quintillion (1 with 18 zeros after it) |
| discrimination became available. These | | | | or more. |
| kits use PCR to amplify specific regions | | | | Y-Chromosome Analysis |
| with known variations and hybridize them | | | | Recent innovations have included the |
| to probes anchored on cards, which | | | | creation of primers targeting |
| results in a colored spot corresponding | | | | polymorphic regions on the Y-chromosome |
| to the particular sequence variation. | | | | (Y-STR), which allows resolution of |
| One of the primary complaints against | | | | multiple male profiles, or cases in |
| RFLP was that it was slow and required | | | | which a differential extraction is not |
| large quantities of DNA to be used. This | | | | possible. Y-chromosomes are paternally |
| led to the development of PCR-based | | | | inherited, so Y-STR analysis can help in |
| methods which required smaller amounts | | | | the identification of paternally related |
| of DNA that could also be more degraded | | | | males. Y-STR analysis was performed in |
| than those used in RFLP analysis. | | | | the Sally Hemings controversy to |
| Systems such as the HLA-DQ alpha reverse | | | | determine if Thomas Jefferson had sired |
| dot blot strips grew to be very popular | | | | a son with one of his slaves. |
| due to their ease of use and the speed | | | | Mitochondrial analysis |
| with which a result could be obtained, | | | | For highly degraded samples, it is |
| however they were not as discriminating | | | | sometimes impossible to get a complete |
| as RFLP. It was also difficult to | | | | profile of the 13 CODIS STRs. In these |
| determine a DNA profile for mixed | | | | situations, mitochondrial DNA (mtDNA) is |
| samples, such as a vaginal swab from a | | | | sometimes typed due to there being many |
| sexual assault victim. | | | | copies of mtDNA in a cell, while there |
| AmpFLP | | | | may only be 1-2 copies of the nuclear |
| Another technique, AmpFLP, or amplified | | | | DNA. Forensic scientists amplify the HV1 |
| fragment length polymorphism was also | | | | and HV2 regions of the mtDNA, then |
| put into practice during the early | | | | sequence each region and compare single |
| 1990's. This technique was also faster | | | | nucleotide differences to a reference. |
| than RFLP analysis and used PCR to | | | | Because mtDNA is maternally inherited, |
| amplify DNA samples. It relied on | | | | directly linked maternal relatives can |
| variable number tandem repeat (VNTR) | | | | be used as match references, such as |
| polymorphisms to distinguish various | | | | one's maternal grandmother's sister's |
| alleles, which were separated on a | | | | son. A difference of two more |
| polyacrylamide gel using an allelic | | | | nucleotides is generally considered to |
| ladder (as opposed to a molecular weight | | | | be an exclusion. Heteroplasmy and poly-C |
| ladder). Bands could be visualized by | | | | differences may throw off straight |
| silver staining the gel. One popular | | | | sequence comparisons, so some expertise |
| locus for fingerprinting was the D1S80 | | | | on the part of the analyst is required. |
| locus. As with all PCR based methods, | | | | mtDNA is useful in determining unclear |
| highly degraded DNA or very small | | | | identities, such as those of missing |
| amounts of DNA may cause allelic dropout | | | | persons when a maternally linked |
| (causing a mistake in thinking a | | | | relative can be found. mtDNA testing was |
| heterozygote is a homozygote) or other | | | | used in determining that Anna Anderson |
| stochastic effects. In addition, because | | | | was not the Russian princess she had |
| the analysis is done on a gel, very high | | | | claimed to be, Anastasia Romanov. |
| number repeats may bunch together at the | | | | mtDNA can be obtained from such material |
| top of the gel, making it difficult to | | | | as hair shafts and old bones/teeth. |
| resolve. AmpFLP analysis can be highly | | | | |