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