Polymerase chain reaction

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Jump to: navigation, search "PCR" redirects here. For other uses, see PCR (disambiguation). Polymerase chain reaction (PCR) is a molecular biology technique [ ! for en"ymatically replicating #$% &ithout using a living organism, such as E. coli or yeast' (ike amplification using living organisms, the technique allo&s a small amount of #$% to be amplified e)ponentially' %s *+, is an in vitro technique, it can be performed &ithout restrictions on the form of #$% and it can be e)tensively modified to perform a &ide array of genetic manipulations' *+, is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and #$% computing' *+, &as invented by -ary .ullis' %t the time he thought up *+, in /01, .ullis &as &orking in 2meryville, +alifornia for +etus, one of the first biotechnology companies' 3here, he &as charged &ith making short chains of #$% for other scientists' .ullis has &ritten that he conceived of *+, &hile cruising along the *acific +oast 4igh&ay one night in his car' 4e &as playing in his mind &ith a ne& &ay of analy"ing changes (mutations) in #$% &hen he reali"ed that he had instead invented a method of amplifying any #$% region' .ullis has said that before his trip &as over, he &as already savoring the prospects of a $obel *ri"e' 4e shared the $obel *ri"e in +hemistry &ith .ichael 5mith in //1' %s .ullis has &ritten in the 5cientific %merican: 67eginning &ith a single molecule of the genetic material #$%, the *+, can generate 88 billion similar molecules in an afternoon' 3he reaction is easy to e)ecute' 9t requires no more than a test tube, a fe& simple reagents, and a source of heat'6

Contents
[hide!

*+, in practice o ' *rimers o ': *rocedure o '1 2)ample o '; *+, optimi"ation o '< #ifficulties &ith polymerase chain reaction '<' *olymerase errors '<': 5i"e limitations

'<'1 $on specific priming '= *ractical modifications to the *+, technique '> ,ecent developments in *+, techniques : ?ses of *+, o :' @enetic fingerprinting o :': *aternity testing o :'1 #etection of hereditary diseases o :'; +loning genes o :'< .utagenesis o :'= %nalysis of ancient #$% o :'> @enotyping of specific mutations o :'0 +omparison of gene e)pression 1 4istory ; *atent &ars < ,eferences o o

= 2)ternal links

[edit] PCR in practice

Figure 1: % thermal cycler for *+, *+, is used to amplify specific regions of a #$% strand' 3his can be a single gene, Aust a part of a gene, or nonBcoding sequence' *+, typically amplifies only short #$% fragments, usually up to 8 kilo base pairs (kb)' +ertain methods can copy fragments up to ;> kb in si"e[citation needed!, &hich is still much less than the chromosomal #$% of a eukaryotic cell B for e)ample, a human cell contains about three billion base pairs' *+,, as currently practiced, requires several basic components' 3hese components are:

DNA tem late, &hich contains the region of the #$% fragment to be amplified 3&o rimers, &hich determine the beginning and end of the region to be amplified (see follo&ing section on primers)

!a" ol#merase (or another durable polymerase), a #$% polymerase, &hich copies the region to be amplified Deo$#nucleotide tri hos hates, (d$3*s) from &hich the #$% polymerase builds the ne& #$% %u&&er, &hich provides a suitable chemical environment for the #$% *olymerase

3he *+, process is carried out in a thermal cycler' 3his is a machine that heats and cools the reaction tubes &ithin it to the precise temperature required for each step of the reaction' 3o prevent evaporation of the reaction mi)ture (typically volumes bet&een <B 88Cl per tube), a heated lid is placed on top of the reaction tubes, or a layer of oil is put on the surface of the reaction mi)ture' 3hese machines cost more than D:,<88 ?5#, as of :88;'

[edit] Primers
3he #$% fragment to be amplified is determined by selecting primers' *rimers are short, artificial #$% strands E often not more than <8 and usually only 0 to :< base pairs long E that are complementary to the beginning or the end of the #$% fragment to be amplified' 3hey anneal by adhering to the #$% template at these starting and ending points, &here the #$% polymerase binds and begins the synthesis of the ne& #$% strand' 3he choice of the length of the primers and their melting temperature (3m) depends on a number of considerations' 3he melting temperature of a primer BB not to be confused &ith the melting temperature of the template #$% BB is defined as the temperature at &hich half of the primer binding sites are occupied' *rimers that are too short &ould anneal at several positions on a long #$% template, &hich &ould result in nonBspecific copies' Fn the other hand, the length of a primer is limited by the ma)imum temperature allo&ed to be applied in order to melt it, as melting temperature increases &ith the length of the primer' .elting temperatures that are too high, i'e', above 08G+, can cause problems since the #$% polymerase is less active at such temperatures' 3he optimum length of a primer is generally from < to ;8 nucleotides &ith a melting temperature bet&een <<G+ and =<G+' 5ometimes degenerate rimers are used' 3hese are actually mi)tures of similar, but not identical, primers' 3hey may be convenient if the same gene is to be amplified from different organisms, as the genes themselves are probably similar but not identical' 3he other use for degenerate primers is &hen primer design is based on protein sequence' %s several different codons can code for one amino acid, it is often difficult to deduce &hich codon is used in a particular case' 3herefore primer sequence corresponding to the amino acid isoleucine might be 6%346, &here % stands for adenine, 3 for thymine, and 4 for adenine, thymine, or cytosine' (5ee genetic code for further details about codons') ?se of degenerate primers can greatly reduce the specificity of the *+, amplification' 3his problem can be partly solved by using touchdo&n *+,'

3he above mentioned considerations make primer design a very e)acting process, upon &hich product yield depends:

@+Bcontent should be bet&een ;8B=8H' +alculated 3m for both primers used in reaction should not differ I<G+, and 3m of the amplification product should not differ from primers by I 8G+' %nnealing temperature usually is <G+ belo& the calculated lo&er 3m' 4o&ever, it should be chosen empirically for individual conditions' 9nner selfBcomplementary hairpins of I; and of dimers I0 should be avoided' *rimer 1J terminus design is critical to *+, success since the primer e)tends from the 1J end' 3he 1J end should not be complementary over greater than 1B; bases to any region of the other primer (or even the same primer) used in the reaction and must provide correct base matching to the template'

3here are computer programs to help design primers (see 2)ternal links)'

[edit] Procedure
3he *+, process usually consists of a series of t&enty to thirtyBfive cycles' 2ach cycle consists of three steps (Fig' :)' ' 3he doubleBstranded #$% has to be heated to /;B/=G+ (or /0G+ if e)tremely thermostable polymerases are used) in order to separate the strands' 3his step is called denaturingK it breaks apart the hydrogen bonds that connect the t&o #$% strands' *rior to the first cycle, the #$% is often denatured for an e)tended time to ensure that both the template #$% and the primers have completely separated and are no& singleBstrand only' 3ime: usually B: minutes, but up to < minutes' %lso certain polymerases are activated at this step (see hotBstart *+,)' :' %fter separating the #$% strands, the temperature is lo&ered so the primers can attach themselves to the single #$% strands' 3his step is called annealing' 3he temperature of this stage depends on the primers and is usually <G+ belo& their melting temperature (;<B=8G+)' % &rong temperature during the annealing step can result in primers not binding to the template #$% at all, or binding at random' 3ime: B: minutes' 1' Finally, the #$% polymerase has to copy the #$% strands' 9t starts at the annealed primer and &orks its &ay along the #$% strand' 3his step is called elongation' 3he elongation temperature depends on the #$% polymerase' 3aq polymerase elongates optimally at a temperature of >: +elsius' 3he time for this step depends both on the #$% polymerase itself and on the length of the #$% fragment to be amplified' %s a ruleBofBthumb, this step takes minute per thousand base pairs' % &inal elongation step is frequently used after the last cycle to ensure that any remaining single stranded #$% is completely copied' 3his differs from all other elongation steps, only in that it is longer, typically 8B < minutes' 3his last step is highly recommendable if the *+, product is to be ligated into a 3 vector using 3%Bcloning'

Figure 2: 5chematic dra&ing of the *+, cycle' ( ) #enaturing at /;B/=G+' (:) %nnealing at (eg) =0G+' (1) 2longation at >:G+ (*L*olymerase)' (;) 3he first cycle is complete' 3he t&o resulting #$% strands make up the template #$% for the ne)t cycle, thus doubling the amount of #$% duplicated for each ne& cycle (a total of three cycles is sho&n above)'

[edit] Example
3he times and temperatures given in this e)ample are taken from a *+, program that &as successfully used on a :<8 bp fragment of the +Bterminus of the insulin'li(e gro)th &actor (*+F)[citation needed!'

@el electrophoresis image of a standard *+,' 3&o sets of specific primers &ere used to amplify one gene from three separate tissues' %s the gel sho&s, 3issue M lacks that gene, &hereas 3issue M: and M1 possess that gene' 3he reaction mi)ture consists of

'8 Cl #$% template ( 88 ngNCl) :'< Cl of primer, ':< Cl per primer ( 88 ngNCl) '8 Cl *fuB*olymerase '8 Cl nucleotides <'8 Cl buffer solution 0/'< Cl &ater

% :88 Cl reaction tube containing the 88 Cl mi)ture is inserted into the thermocycler' 3he *+, process consists of the follo&ing steps: ' 9nitiali"ation' 3he mi)ture is heated at /=G+ for < minutes to ensure that the #$% strands as &ell as the primers have melted' 3he #$% *olymerase can be present at initiali"ation, or it can be added after this step' :' .elting, &here it is heated at /=G+ for 18 seconds' For each cycle, this is usually enough time for the #$% to denature' 1' %nnealing by heating at =0G+ for 18 seconds:3he primers are Aiggling around, caused by the 7ro&nian motion' 5hort bondings are constantly formed and broken bet&een the single stranded primer and the single stranded template' 3he more stable bonds last a little bit longer (primers that fit e)actly) and on that little piece of double stranded #$% (template and primer), the polymerase can attach and starts copying the template' Fnce there are a fe& bases built in, the 3m of the doubleBstranded region bet&een the template and the primer is greater than the annealing or e)tension temperature'

;' 2longation by heating >:G+ for ;< seconds:3his is the ideal &orking temperature for the polymerase' 3he primers, having been e)tended for a fe& bases, already have a stronger hydrogen bond to the template than the forces breaking these attractions' *rimers that are on positions &ith no e)act match, melt a&ay from the template (because of the higher temperature) and are not e)tended' 3he bases (complementary to the template) are coupled to the primer on the 1J side (the polymerase adds d$3*Js from <J to 1J, reading the template from 1J to <J side, bases are added complementary to the template) ' 5teps :B; are repeated :< times, but &ith good primers and fresh polymerase, < to :8 cycles is sufficient' :' .i)ture is held at >G+' 3his is useful if one starts the *+, in the evening Aust before leaving the lab, so it can run overnight' 3he #$% &ill not be damaged at >G+ after Aust one night' 3he *+, product can be identified by its si"e using agarose gel electrophoresis' %garose gel electrophoresis is a procedure that consists of inAecting #$% into agarose gel and then applying an electric current to the gel' %s a result, the smaller #$% strands move faster than the larger strands through the gel to&ard the positive current' 3he si"e of the *+, product can be determined by comparing it &ith a DNA ladder, &hich contains #$% fragments of kno&n si"e, also &ithin the gel (Fig' 1)'

[edit] PCR optimization

5ince *+, is very sensitive, adequate measures to avoid contamination from other #$% present in the lab environment (bacteria, viruses, lab staffJs skin etc') should be taken' 3hus #$% sample preparation, reaction mi)ture assemblage and the *+, process, in addition to the subsequent reaction product analysis, should be performed in separate areas' For the preparation of reaction mi)ture, a laminar flo& cabinet &ith ?O lamp is recommended' Fresh gloves should be used for each *+, step as &ell as displacement pipettes &ith aerosol filters' 3he reagents for *+, should be prepared separately and used solely for this purpose' %liquots should be stored separately from other #$% samples' % control reaction (inner control), omitting template #$%, should al&ays be performed, to confirm the absence of contamination or primer multimer formation'

[edit] Difficulties ith polymerase chain reaction

*olymerase chain reaction is not perfect, and errors and mistakes can occur' 3hese are some common errors and problems that may occur' [edit] Polymerase errors 3aq polymerase lacks a 1J to <J e)onuclease activity' 3his makes it impossible for it to check the base it has inserted and remove it if it is incorrect, a process common in higher

organisms' 3his in turn results in a high error rate of appro)imately in 8,888 bases, &hich, if an error occurs early, can alter large proportions of the final product' Fther polymerases are available for accuracy in vital uses such as amplification for sequencing' 2)amples of polymerases &ith 1Jto <J e)onuclease activity include: -F# #$% polymerase, a recombinant form of !hermococcus (oda(araensis -F# K Oent, &hich is e)tracted from !hermococcus litoralisK *fu #$% polymerase, &hich is e)tracted from P#rococcus &uriosusK and *&o, &hich is e)tracted from P#rococcus )oesii' [edit] !ize limitations *+, &orks readily &ith #$% of lengths t&o to three thousand basepairs, but above this length the polymerase tends to fall off, and the typical heating cycle does not leave enough time for polymerisation to complete' 9t is possible to amplify larger pieces of up to <8,888 base pairs &ith a slo&er heating cycle and special polymerases' 3hese special polymerases are often polymerases fused to a #$%Bbinding protein, making them literally 6stick6 to the #$% longer' [edit] "on specific priming 3he non specific binding of primers is al&ays a possibility due to sequence duplications, nonBspecific binding and partial primer binding, leaving the <J end unattached' 3his is increased by the use of degenerate sequences or bases in the primer' .anipulation of annealing temperature and magnesium ion (&hich stabilise #$% and ,$% interactions) concentrations can increase specificity' $onBspecific priming can be prevented during the lo& temperatures of reaction preparation by use of 6hotBstart6 polymerase en"ymes &here the active site is blocked by an antibody or chemical that only dislodges once the reaction is heated to /<P+ during the denaturation step of the first cycle' Fther methods to increase specificity include $ested *+, and 3ouchdo&n *+,'

[edit] Practical modifications to the PCR techni#ue

"ested PCR B $ested *+, is intended to reduce the contaminations in products due to the amplification of une)pected primer binding sites' 3&o sets of primers are used in t&o successive *+, runs, the second set intended to amplify a secondary target &ithin the first run product' 3his is very successful, but requires more detailed kno&ledge of the sequences involved' $nterse#uence specific %$!!R& PCR 'igation(mediated PCR $n)erse PCR B 9nverse *+, is a method used to allo& *+, &hen only one internal sequence is kno&n' 3his is especially useful in identifying flanking sequences to various genomic inserts' 3his involves a series of digestions and self ligation before cutting by an endonuclease, resulting in kno&n sequences at either end of the unkno&n sequence'

R*(PCR B ,3B*+, (Reverse *ranscription *+,) is the method used to amplify, isolate or identify a kno&n sequence from a cell or tissues ,$% library' 2ssentially normal *+, preceded by transcription by ,everse transcriptase (to convert the ,$% to c#$%) this is &idely used in e)pression mapping, determining &hen and &here certain genes are e)pressed' +ssem,ly PCR B %ssembly *+, is the completely artificial synthesis of long gene products by performing *+, on a pool of long oligonucleotides &ith short overlapping segments' 3he oligonucleotides alternate bet&een sense and antisense directions, and the overlapping segments serve to order the *+, fragments so that they selectively produce their final product' +symmetric PCR B %symmetric *+, is used to preferentially amplify one strand of the original #$% more than the other' 9t finds use in some types of sequencing and hybridi"ation probing &here having only one of the t&o complementary stands is ideal' *+, is carried out as usual, but &ith a great e)cess of the primers for the chosen strand' #ue to the slo& (arithmetic) amplification later in the reaction after the limiting primer has been used up, e)tra cycles of *+, are required' % recent modification on this process, kno&n as 'inearB+fterB*heB E)ponentialB*+, ((%32B*+,), uses a limiting primer &ith a higher melting temperature (3m) than the e)cess primer to maintain reaction efficiency as the limiting primer concentration decreases midBreaction' -uantitati)e PCR B QB*+, (-uantitative *+,) is used to rapidly measure the quantity of *+, product (preferably realBtime), thus is an indirect method for quantitatively measuring starting amounts of #$%, c#$% or ,$%' 3his is commonly used for the purpose of determining &hether a sequence is present or not, and if it is present the number of copies in the sample' 3here are 1 main methods &hich vary in difficulty and detail' -uantitati)e real(time PCR is often confusingly kno&n as ,3B*+, (Real *ime *+,) and ,QB*+,' Q,3B*+, or ,3QB*+, are more appropriate contractions' ,3B*+, can also refer to reverse transcription *+,, &hich even more confusingly, is often used in conAunction &ith QB*+,' 3his method uses fluorescent dyes and probes to measure the amount of amplified product in real time' *ouchdo n PCR B 3ouchdo&n *+, is a variant of *+, that reduces nonspecific primer annealing by more gradually lo&ering the annealing temperature bet&een cycles' %s higher temperatures give greater specificity for primer binding, primers anneal first as the temperature passes through the "one of greatest specificity' .ot(start PCR is a technique that reduces nonBspecific priming that occurs during the preparation of the reaction components' 3he technique may be performed manually by simply heating the reaction components briefly at the melting temperature before adding the polymerase' 5peciali"ed en"yme systems

have been developed that inhibit the polymeraseJs activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a highBtemperature activation step'

Colony PCR B 7acterial clones (2'coli) can be screened for the correct ligation products' 5elected colonies are picked &ith a sterile toothpick from an agarose plate and dabbed into the master mi) or sterile &ater' *rimers (and the master mi)) are added B the *+, protocol has to be started &ith an e)tended time at /<RR+' R+CE(PCR B ,apid amplification of c#$% ends' /ultiplex(PCR B 3he use of multiple, unique primer sets &ithin a single *+, reaction to produce amplicons of varying si"es specific to different #$% sequences' 7y targeting multiple genes at once, additional information may be elicited from a single test run that other&ise &ould require several times the reagents and technician time to perform' %nnealing temperatures for each of the primer sets must be optimi"ed to &ork correctly &ithin a single reaction and amplicon si"es should be separated by enough difference in final base pair length to form distinct bands via gel electrophoresis' /ethylation !pecific PCR B .ethylation 5pecific *+, (.5*) is used to detect methylation of +p@ islands in genomic #$%' #$% is first treated &ith sodium bisulfite, &hich converts unmethylated cytosine bases to uracil, &hich is recogni"ed by *+, primers as thymine' 3&o *+, reactions are then carried out on the modified #$%, using primer sets identical e)cept at any +p@ islands &ithin the primer sequences' %t these points, one primer set recogni"es #$% &ith cytosines to amplify methylated #$%, and one set recogni"es #$% &ith uracil or thymine to amplify unmethylated #$%' .5* using q*+, can also be performed to obtain quantitative rather than qualitative information about methylation'

[edit] Recent de)elopments in PCR techni#ues

% more recent method &hich e)cludes a temperature cycle, but uses en"ymes, is helicaseBdependent amplification' 3%9(B*+,, developed by (iu et al. in //<, is the thermal asymmetric interlaced *+,' .etaB*+,, developed by %ndre& Wallace, allo&s to optimi"e amplification and direct sequence analysis of comple) genes' #etails at $ational @enetic ,eference (aboratory, .anchester, ?-

[edit] 0ses of PCR

*+, can be used for a broad variety of e)periments and analyses' 5ome e)amples are discussed belo&'

[edit] 1enetic fingerprinting

@enetic fingerprinting is a forensic technique used to identify a person by comparing his or her #$% &ith a given sample' %n e)ample is blood from a crime scene being genetically compared to blood from a suspect' 3he sample may contain only a tiny amount of #$% (obtained from a source such as blood, semen, saliva, hair, or other organic material))' 3heoretically, Aust a single strand is needed' First, one breaks the #$% sample into fragmentsK then amplifies them using *+,' 3he amplified fragments are then separated using gel electrophoresis' 3he overall layout of the #$% fragments is called a DNA &inger rint' 5ince there is a very tiny possibility that t&o individuals may have the same sequences (one in several million), the technique is more effective at acquitting a suspect than proving the suspect guilty' 3his small possibility &as e)ploited by defense la&yers in the controversial F'J' 5impson case' % match ho&ever usually remains an e)tremely strong indicator also in the question of guilt'

[edit] Paternity testing

Figure 2: 2lectrophoresis of *+,Bamplified #$% fragments' ( ) Father' (:) +hild' (1) .other' 3he child has inherited some, but not all of the fingerprint of each of its parents, giving it a ne&, unique fingerprint' %lthough these resulting JfingerprintsJ are unique (e)cept for identical t&ins), genetic relationships, for e)ample, parentBchild or siblings, can be determined from t&o or more genetic fingerprints, &hich can be used for paternity tests (Fig' ;)' % variation of this technique can also be used to determine evolutionary relationships bet&een organisms'

[edit] Detection of hereditary diseases

3he detection of hereditary diseases in a given genome is a long and difficult process, &hich can be shortened significantly by using *+,' 2ach gene in question can easily be amplified through *+, by using the appropriate primers and then sequenced to detect mutations' Oiral diseases, too, can be detected using *+, through amplification of the viral #$%' 3his analysis is possible right after infection, &hich can be from several days to several months before actual symptoms occur' 5uch early diagnoses give physicians a significant lead in treatment'

[edit] Cloning genes

+loning a gene, not to be confused &ith cloning a &hole organism, describes the process of isolating a gene from one organism and then inserting it into another organism (no& termed a genetically modified organism (@.F))' *+, is often used to amplify the gene, &hich can then be inserted into a vector (a vector is a piece of #$% &hich JcarriesJ the gene into the @.F) such as a plasmid (a circular #$% molecule) (Fig' <)' 3he #$% can then be transferred into an organism (the @.F) &here the gene and its product can be studied more closely' 2)pressing a cloned gene (&hen a gene is e$ ressed the gene product (usually protein or ,$%) is produced by the @.F) can also be a &ay of massB producing useful proteins, for e)ample medicines or the en"ymes in biological &ashing po&ders' 3he incorporation of an affinity tag on a recombinant protein &ill generate a fusion protein &hich can be more easily purified by affinity chromatography'

Figure 3: +loning a gene using a plasmid' ( ) +hromosomal #$% of organism %' (:) *+,' (1) .ultiple copies of a single gene from organism %' (;) 9nsertion of the gene into a plasmid' (<) *lasmid &ith gene from organism %' (=) 9nsertion of the plasmid in organism 7' (>) .ultiplication or e)pression of the gene, originally from organism %, occurring in organism 7'

[edit] /utagenesis
,utagenesis is a &ay of making changes to the sequence of nucleotides in the #$%' 3here are situations in &hich one is interested in mutated (changed) copies of a given #$% strand, for e)ample, &hen trying to assess the function of a gene or in in'vitro protein evolution (also kno&n as #irected evolution)' .utations can be introduced into copied #$% sequences in t&o fundamentally different &ays in the *+, process' -ite'

directed mutagenesis allo&s the e)perimenter to introduce a mutation at a specific location on the #$% strand' ?sually, the desired mutation is incorporated in the primers used for the *+, program' Random mutagenesis, on the other hand, is based on the use of errorBprone polymerases in the *+, process' 9n the case of random mutagenesis, the location and nature of the mutations cannot be controlled' Fne application of random mutagenesis is to analy"e structureBfunction relationships of a protein' 7y randomly altering a #$% sequence, one can compare the resulting protein &ith the original and determine the function of each part of the protein'

[edit] +nalysis of ancient D"+

?sing *+,, it becomes possible to analy"e #$% that is thousands of years old' *+, techniques have been successfully used on animals, such as a fortyBthousandByearBold mammoth, and also on human #$%, in applications ranging from the analysis of 2gyptian mummies to the identification of a ,ussian 3sar'

[edit] 1enotyping of specific mutations

3hrough the use of alleleBspecific *+,, one can easily determine &hich allele of a mutation or polymorphism an individual has' 4ere, one of the t&o primers is common, and &ould anneal a short distance a&ay from the mutation, &hile the other anneals right on the variation' 3he 1J end of the alleleBspecific primer is modified, to only anneal if it matches one of the alleles' 9f the mutation of interest is a 3 or + single nucleotide polymorphism (3N+ 5$*), one &ould use t&o reactions, one containing a primer ending in 3, and the other ending in +' 3he common primer &ould be the same' Follo&ing *+,, these t&o sets of reactions &ould be run out on an agarose gel, and the band pattern &ill tell you if the individual is homo"ygous 3, homo"ygous +, or hetero"ygous 3N+' 3his methodology has several applications, such as amplifying certain haplotypes (&hen certain alleles at : or more 5$*s occur together on the same chromosome (inkage #isequilibrium) or detection of recombinant chromosomes and the study of meiotic recombination'

[edit] Comparison of gene expression

,esearchers have used traditional *+, as a &ay to estimate changes in the amount of a geneJs e)pression' ,ibonucleic acid (,$%) is the molecule into &hich #$% is transcribed prior to making a protein, and those strands of ,$% that hold the instructions for protein sequence are kno&n as messenger ,$% (m,$%)' Fnce ,$% is isolated it can be reverse transcribed back into #$% (complementary #$% to be precise, kno&n as c#$%), at &hich point traditional *+, can be applied to amplify the gene, this methodology is called ,3B*+,' 9n most cases if there is more starting material (m,$%) of a gene then during *+, more copies of the gene &ill be generated' When the products of the *+, process are run on an agarose gel (see Figure 1 above) a band, corresponding to a gene, &ill appear larger on the gel (note that the band remains in the same location relative to the ladder, it &ill Aust appear fatter or brighter)' 7y running samples of amplified c#$% from differently treated organisms one can get a general idea of &hich

sample e)pressed more of the gene of interest' % quantative ,3B*+, method has been developed, it is called ,ealBtime *+, '

[edit] .istory
*olymerase chain reaction &as invented by -ary .ullis' 4e &as a&arded the $obel *ri"e in +hemistry in //1 for his invention, only seven years after he and his colleagues at +etus first reduced his proposal to practice' 3he idea &as to develop a process by &hich #$% could be artificially multiplied through repeated cycles of duplication driven by an en"yme called #$% polymerase' #$% polymerase occurs naturally in living organisms' 9n cells it functions to duplicate #$% &hen cells divide in mitosis and meiosis' *olymerase &orks by binding to a single #$% strand and creating the complementary strand' 9n the first of many original processes, the en"yme &as used in vitro (in a controlled environment outside an organism)' 3he doubleBstranded #$% &as separated into t&o single strands by heating it to /;G+ (:8 GF)' %t this temperature, ho&ever, the #$% polymerase used at the time &ere destroyed, so the en"yme had to be replenished after the heating stage of each cycle' 3he original procedure &as very inefficient, since it required a great deal of time, large amounts of #$% polymerase, and continual attention throughout the process' (ater, this original *+, process &as greatly improved by the use of #$% polymerase taken from thermophilic bacteria gro&n in geysers at a temperature of over 8G+ (:18GF)' 3he #$% polymerase taken from these organisms is stable at high temperatures and, &hen used in *+,, does not break do&n &hen the mi)ture &as heated to separate the #$% strands' 5ince there &as no longer a need to add ne& #$% polymerase for each cycle, the process of copying a given #$% strand could be simplified and automated' Fne of the first thermostable #$% polymerases &as obtained from !hermus a"uaticus and &as called 63aq'6 3aq polymerase is &idely used in current *+, practice' % disadvantage of 3aq is that it sometimes makes mistakes &hen copying #$%, leading to mutations (errors) in the #$% sequence, since it lacks 1JS<J proofreading e)onuclease activity' *olymerases such as P)o or P&u, obtained from Archaea, have roo&reading mechanisms (mechanisms that check for errors) and can significantly reduce the number of mutations that occur in the copied #$% sequence' 4o&ever these en"ymes polymerise #$% at a much slo&er rate than 3aq' +ombinations of both !a" and P&u are available no&adays that provide both high processivity (fast polymerisation) and high fidelity (accurate duplication of #$%)' *+, has been performed on #$% larger than 8 kilobases, but the average *+, is only several hundred to a fe& thousand bases of #$%' 3he problem &ith long *+, is that there is a balance bet&een accuracy and processivity of the en"yme' ?sually, the longer the fragment, the greater the probability of errors'

[edit] Patent ars

3he *+, technique &as patented by +etus +orporation, &here .ullis &orked &hen he invented the technique in /01' 3he 3aq polymerase en"yme is also covered by patents' 3here have been several highBprofile la&suits related to the technique, including an unsuccessful la&suit brought by #u*ont' 3he pharmaceutical company 4offmannB(a ,oche purchased the rights to the patents in //: and currently holds those that are still protected' % related patent battle over the 3aq polymerase en"yme is still ongoing in several Aurisdictions around the &orld bet&een ,oche and *romega' 9nterestingly, it seems possible that the legal arguments &ill e)tend beyond the life of the original *+, and 3aq polymerase patents, &hich e)pire in :88='

[edit] References
' 4 3he history of *+,: 5mithsonian 9nstitution %rchives, 9nstitutional 4istory #ivision' ,etrieved :; June :88='

5ambrook, Joseph, and David .. Russell (/001). ,olecular Cloning2 A 3aborator# ,anual, 1rd ed', +old 5pring 4arbor, $'T': +old 5pring 4arbor (aboratory *ress' 957$ 8B0>/=/B<>=B<. .ullis, -ary (1445). Dancing Na(ed in the ,ind Field' $e& Tork: *antheon 7ooks' 957$ 8B=>/B;;:<<B1. ,abino&, *aul (1446). ,a(ing PCR2 A -tor# o& %iotechnolog#' +hicago: ?niversity

1enetic disorder
From Wikipedia, the free encyclopedia

(,edirected from 4ereditary disease) Jump to: navigation, search % genetic disorder, or genetic disease, is a disease caused by abnormal e)pression of one or more genes in a person causing a clinical phenotype' 5ome conditions labelled a disorder may confer an advantage' 3here are a number of possible path&ays of genetic defects, the simplest of &hich are summari"ed thus:

3here are genetic disorders caused by the abnormal chromosome number, as in #o&n syndrome (e)tra chromosome : ) and -linefelterJs syndrome (a male &ith : U chromosomes)' 3riplet e)pansion repeat mutations can cause Fragile U syndrome or 4untingtonJs disease, by modification of gene e)pression or gain of function, respectively' #efective genes are often inherited from the parents' 9n this case, the genetic disorder is kno&n as a hereditary disease' 3his can often happen une)pectedly

&hen t&o healthy carriers of a defective recessive gene reproduce, but can also happen &hen the defective gene is dominant' +urrently around ;,888 genetic disorders are kno&nK ne& ones are constantly being discovered' 3he vast maAority of these disorders are quite rare, and affect one person in every several thousands or millions' +ystic fibrosis is one of the most common genetic disordersK around <H of the population of the ?nited 5tates carry at least one copy of the defective gene' %dditionally, some people in Japan developed genetic disorders' 3his &as in part due to the effects of radiation from the atomic bombs dropped on 4iroshima and $agasaki'

Contents
[hide!

5ingle gene disorders o ' 3ransmission of single gene disorders : .ultifactorial and polygenic disorders 1 +hromosomal disorders ; 5tudy of @enetic #iseases < .edical diagnosis, treatment, and counseling = 5ee also > ,eferences 0 2)ternal links

[edit] !ingle gene disorders

[edit] *ransmission of single gene disorders
Where genetic disorders are the result of a single mutated gene they can be passed on to subsequent generations in the &ays outlined in the table belo&' @enomic imprinting and uniparental disomy, ho&ever, may affect inheritance patterns' 3he divisions bet&een recessive and dominant are not 6hard and fast6 although the divisions bet&een autosomal and UBlinked are (related to the position of the gene)' For e)ample, achondroplasia is typically considered a dominant disorder, but young goats or children &ith t&o genes for achondroplasia have a severe skeletal disorder that achondroplasics could be vie&ed as carriers of' 5ickleBcell anemia is also considered a recessive condition, but carriers that have it by half along &ith the normal gene have increased immunity to malaria in early childhood, &hich could be described as a related dominant condition' Examples 5historymatters5org5u6

$nheritance pattern

Description

%utosomal dominant

Fnly one mutated copy of the gene is needed for a person to be affected by an autosomal dominant disorder' 2ach affected person usually has one affected parent' 3here is a <8H chance that a child &ill inherit the 4untingtons disease, mutated gene' +onditions that are $eurofibromatosis , 47F+ autosomal dominant have lo& syndrome, 4ereditary penetrance, &hich means that, nonpolyposis colorectal cancer although only one mutated copy is needed, a relatively small proportion of those &ho inherit that mutation go on to develop the disease, often later in life' 3&o copies of the gene must be mutated for a person to be affected by an autosomal recessive disorder' %n affected person usually has unaffected parents &ho each carry a single copy of the mutated gene (and are referred to as carriers)' 3&o unaffected people &ho each carry one copy of the mutated gene have a :<H chance &ith each pregnancy of having a child affected by the disorder'

%utosomal recessive

+ystic fibrosis, 5ickle cell anemia, 3ayB5achs disease, 5pinal muscular atrophy, .uscular dystrophy

UBlinked dominant

UBlinked dominant disorders are caused by mutations in genes on the U chromosome' Fnly a fe& disorders have this inheritance pattern' Females are more frequently affected than males, and the chance of passing on an UBlinked dominant disorder differs bet&een men and &omen' 3he sons of a man &ith an UBlinked dominant 4ypophosphatemia, %icardi disorder &ill not be affected, and his 5yndrome daughters &ill all inherit the condition' % &oman &ith an UBlinked dominant disorder has a <8H chance of having an affected daughter or son &ith each pregnancy' 5ome UBlinked dominant conditions, such as %icardi 5yndrome, are fatal to boys, therefore only girls have them (and boys &ith -linefelter 5yndrome)' UBlinked recessive disorders are also 4emophilia %, #uchenne muscular

UBlinked

recessive

caused by mutations in genes on the U chromosome' .ales are more frequently affected than females, and the chance of passing on the disorder differs bet&een men and &omen' 3he sons of a man &ith an UBlinked recessive disorder &ill not be dystrophy, +olor blindness, 3urner affected, and his daughters &ill carry 5yndrome one copy of the mutated gene' With each pregnancy, a &oman &ho carries an UBlinked recessive disorder has a <8H chance of having sons &ho are affected and a <8H chance of having daughters &ho carry one copy of the mutated gene' TBlinked disorders are caused by mutations on the T chromosome' Fnly males can get them, and all of the sons of an affected father are affected' 5ince the T chromosome is .ale 9nfertility very small, TBlinked disorders only cause infertility, and may be circumvented &ith the help of some fertility treatments'

TBlinked

3his type of inheritance, also kno&n as maternal inheritance, applies to genes in mitochondrial #$%' 7ecause only egg cells contribute .itochondrial mitochondria to the developing embryo, only females can pass on mitochondrial conditions to their children'

(eberJs 4ereditary Fptic $europathy ((4F$)

[edit] /ultifactorial and polygenic disorders

@enetic disorders may also be comple), multifactorial or polygenic, this means that they are likely associated &ith the effects of multiple genes in combination &ith lifestyle and environmental factors' .ultifactoral disorders include heart disease and diabetes' %lthough comple) disorders often cluster in families, they do not have a clearBcut pattern of inheritance' 3his makes it difficult to determine a personVs risk of inheriting or passing on these disorders' +omple) disorders are also difficult to study and treat because the specific factors that cause most of these disorders have not yet been identified' Fn a pedigree, polygenic diseases do tend to Wrun in familiesX, but the inheritance does not fit simple patterns as &ith .endelian diseases' 7ut this does not mean that the genes

cannot eventually be located and studied' 3here is also a strong environmental component to many of them (e'g', blood pressure)' 2)amples of polygenic disorders in humans include:

%utism 4eart disease hypertension diabetes obesity cancers cleft palate (and many more)

[edit] Chromosomal disorders

,ain article2 Chromosome abnormalities +hanges that affect entire chromosomes or segments of chromosomes can cause problems &ith gro&th, development, and function of the bodyJs systems' 3hese changes can affect many genes along the chromosome and alter the proteins made by those genes' +onditions caused by a change in the number or structure of chromosomes are kno&n as chromosomal disorders' 5ome chromosomal conditions are caused by changes in the number of chromosomes, called aneuploidy' 3hese changes are not inherited, but occur as random events during the formation of reproductive cells (ova and sperm cells)' %n error in cell division called nondisAunction results in reproductive cells &ith an abnormal number of chromosomes' For e)ample, a reproductive cell may accidentally gain or lose one copy of a chromosome' 9f one of these atypical reproductive cells contributes to the genetic makeup of a child, the child &ill have an e)tra (trisomy) or missing chromosome (monosomy) in each of the bodyVs cells' 3he formation of ring chromosomes follo&ing fertili"ation also cause genetic disorders' +hromosomal disorders can also be caused by chromosome structure' 3hese changes are caused by the breakage and reunion of chromosome segments &hen an egg or sperm cell is formed or in early fetal development' *ieces of #$% can be rearranged &ithin one chromosome, or transferred bet&een t&o or more chromosomes' 3he effects of structural changes depend on their si"e and location' .any different structural changes are possibleK some cause medical problems, &hile others may have no effect on a personVs health' %lthough it is possible to inherit some types of chromosomal abnormalities, most chromosomal disorders are not passed from one generation to the ne)t'

[edit] !tudy of 1enetic Diseases

3he study of genetic diseases is a large scientific discipline, &hose theoretical underpinning is based on *opulation genetics'

[edit] /edical diagnosis7 treatment7 and counseling

@enetic diseases are typically diagnosed and treated by geneticists' @enetic counselors assist the physicians and directly counsel patients'

[edit] !ee also

(ist of genetic disorders

[edit] References
!his article incor orates ublic domain te$t &rom !he 7.-. National 3ibrar# o& ,edicine

1enetic fingerprinting
From Wikipedia, the free encyclopedia

(,edirected from @enetic fingerprint) Jump to: navigation, search 1enetic fingerprinting, D"+ testing, D"+ typing, and D"+ profiling are techniques used to distinguish bet&een individuals of the same species using only samples of their #$%' 9ts invention by 5ir %lec Jeffreys at the ?niversity of (eicester &as announced in /0<' 3&o humans &ill have the vast maAority of their #$% sequence in common' @enetic fingerprinting e)ploits highly variable repeating sequences called minisatellites' 3&o unrelated humans &ill be likely to have different numbers of minisatellites at a given locus' 7y using *+, enough #$% is obtained to then detect the number of repeats at several loci' 9t is possible to establish a match that is e)tremely unlikely to have arisen by coincidence, e)cept in the case of identical t&ins, &ho &ill have identical genetic profiles' @enetic fingerprinting is used in forensic science, to match suspects to samples of blood, hair, saliva or semen' 9t has also led to several e)onerations of formerly convicted suspects' 9t is also used in such applications as identifying human remains, paternity testing, match organ donors, studying populations of &ild animals, and establishing the province or composition of foods' 9t has also been used to generate hypotheses on the pattern of the human diaspora in prehistoric times' 3esting is subAect to the legal code of the Aurisdiction in &hich it is performed' ?sually the testing is voluntary, but it can be made compulsory by such instruments as a search

&arrant or court order' 5everal Aurisdictions have also begun to assemble databases containing #$% information of convicts' 3he ?nited -ingdom currently has the most e)tensive #$% database in the &orld, &ith &ell over : million records as of :88<: 3he $ational #$% #atabase ($#$%#)' 3he si"e of this database, and its rate of gro&th, is giving concern to civil liberties groups in the ?-, &here police have &ideBranging po&ers to take samples and retain them even in the event of acquittal'

Contents
[hide!

,eference samples : #$% fingerprinting methods o :' ,F(* analysis o :': *+, analysis o :'1 %mpF(* o :'; 53, analysis o :'< TB+hromosome %nalysis o :'= .itochondrial analysis 1 +onsiderations &hen evaluating #$% evidence ; Fake #$% evidence < +ases = 5ee also > 2)ternal links

[edit] Reference samples

#$% identification must be done by comparing the remains to reference samples, &hich can come from a number of sources:

*ersonal items (e'g' toothbrush, ra"or, ''') 7anked samples (e'g' banked sperm or biopsy tissue) 7lood kin (biological relative) 4uman remains previously identified

,eference samples are often collected using buccal s&ab'

[edit] D"+ fingerprinting methods

#$% fingerprinting begins by e)tracting #$% from the cells in a sample of blood, saliva, semen, or other appropriate fluid or tissue'

[edit] RF'P analysis

When #$% fingerprinting first began, restriction fragment length polymorphism (,F(*) analysis &as used, though it has been almost completely replaced &ith ne&er techniques' ,F(* analysis is performed by using a restriction en"yme to cut the #$% into fragments &hich are separated into bands during agarose gel electrophoresis' $e)t, the bands of #$% are transferred via a technique called 5outhern blotting from the agarose gel to a nylon membrane' 3his is treated &ith a radioactivelyBlabelled #$% probe &hich binds to certain specific #$% sequences on the membrane' 3he e)cess #$% probe is then &ashed off' %n UBray film placed ne)t to the nylon membrane detects the radioactive pattern' 3his film is then developed to make a visible pattern of bands called a #$% fingerprint' 7y using multiple probes targeting various polymorphisms in successive UBray images, a fairly high degree of discrimination &as possible' 3he primary dra&back of ,F(* is that the e)act si"es of the bands are unkno&n and comparison to a molecular &eight ladder is done in a purely qualitative manner' .any labs developed policies that described &hat they considered a unique band, but it &as not standardi"ed and led to #$% fingerprinting coming under harsh attack in *eople v' +astro <;< $'T'5' :d' /0< (5up' +t' /0/)' ,F(* &as a very time consuming method &hich required relatively high quantity of good quality #$% to be used (such as a dime si"ed blood drop)' 3his made typing degraded samples such as those from evidence that had been e)posed to the elements fairly difficult'

[edit] PCR analysis

With the invention of polymerase chain reaction (*+,), #$% fingerprinting took huge strides for&ard in both discriminating po&er and ability to recover information from very small starting samples' *+, involves the amplification of specific regions of #$% using a cycling of temperature and a thermostable polymerase en"yme along &ith sequence specific primers of #$%' +ommercial kits that used single nucleotide polymorphisms (5$*s) for discrimination became available' 3hese kits use *+, to amplify specific regions &ith kno&n variations and hybridi"e them to probes anchored on cards, &hich results in a colored spot corresponding to the particular sequence variation' Fne of the primary complaints against ,F(* &as that it &as slo& and required large quantities of #$% to be used' 3his led to the development of *+,Bbased methods &hich required smaller amounts of #$% that could also be more degraded than those used in ,F(* analysis' 5ystems such as the 4(%B#Q alpha reverse dot blot strips gre& to be very popular due to their ease of use and the speed &ith &hich a result could be obtained, ho&ever they &ere not as discriminating as ,F(*' 9t &as also difficult to determine a #$% profile for mi)ed samples, such as a vaginal s&ab from a se)ual assault victim'

[edit] +mpF'P
%nother technique, %mpF(*, or amplified fragment length polymorphism &as also put into practice during the early //8Js' 3his technique &as also faster than ,F(* analysis and used *+, to amplify #$% samples' 9t relied on variable number tandem repeat

(O$3,) polymorphisms to distinguish various alleles, &hich &ere separated on a polyacrylamide gel using an allelic ladder (as opposed to a molecular &eight ladder)' 7ands could be visuali"ed by silver staining the gel' Fne popular locus for fingerprinting &as the # 508 locus' %s &ith all *+, based methods, highly degraded #$% or very small amounts of #$% may cause allelic dropout (causing a mistake in thinking a hetero"ygote is a homo"ygote) or other stochastic effects' 9n addition, because the analysis is done on a gel, very high number repeats may bunch together at the top of the gel, making it difficult to resolve' %mpF(* analysis can be highly automated, and allo&s for easy creation of phylogenetic trees based on comparing individual samples of #$%' #ue to its relatively lo& cost and ease of setBup and operation, %mpF(* remains popular in lo&er income countries'

[edit] !*R analysis

3he most prevalent method of #$% fingerprinting used today is based on *+, and uses short tandem repeats (53,)' 3his method uses highly polymorphic regions that have short repeated sequences of #$% (the most common is ; bases repeated, but there are lengths in use, including 1 and < bases)' 7ecause different people have different numbers of repeat units, these regions of #$% can be used to discriminate bet&een individuals' 3hese 53, loci (locations) are targeted &ith sequenceBspecific primers and are amplified using *+,' 3he #$% fragments that result are then separated and detected using electrophoresis' 3here are t&o common methods of separation and detection, capillary electrophoresis (+2) and gel electrophoresis' 3he polymorphisms displayed at each 53, region are by themselves very common, typically each polymorphism &ill be shared by around < B :8H of individuals' When looking at multiple loci, it is the unique combinations of these polymorphisms to an individual that makes this method discriminating as an identification tool' 3he more 53, regions that are tested in an individual the more discriminating the test becomes' From country to country different 53, based #$% profiling systems are in use' 9n $orth %merica +F#95 is prevalent, &hile in the ?- the 5@.Y system, &hich is compatible &ith 3he $ational #$% #atabase is in use' Whichever system is used, many of the 53, regions under test are the same' 3hese #$% profiling systems are based around multiple) reactions, &hereby many 53, regions &ill be under test at the same time' +apillary electrophoresis &orks by electrokinetically (movement through the application of an electric field) inAecting the #$% fragments into a thin glass tube (the capillary) filled &ith polymer' 3he #$% is pulled through the tube by the application of an electric field, separating the fragments such that the smaller fragments travel faster through the capillary' 3he fragments are then detected using fluorescent dyes that &ere attached to the primers used in *+,' 3his allo&s multiple fragments to be amplified and run simultaneously, something kno&n as multiple)ing' 5i"es are assigned using labeled #$% si"e standards that are added to each sample, and the number of repeats are determined by comparing the si"e to an allelic ladder, a sample that contains all of the common possible repeat si"es' %lthough this method is e)pensive, larger capacity machines &ith higher

throughput are being used to lo&er the costNsample and reduce backlogs that e)ist in many government crime facilities' @el electrophoresis acts using similar principles as +2, but instead of using a capillary, a large polyacrylamide gel is used to separate the #$% fragments' %n electric field is applied, as in +2, but instead of running all of the samples by a detector, the smallest fragments are run close to the bottom of the gel and the entire gel is scanned into a computer' 3his produces an image sho&ing all of the bands corresponding to different repeat si"es and the allelic ladder' 3his approach does not require the use of si"e standards, since the allelic ladder is run alongside the samples and serves this purpose' Oisuali"ation can either be through the use of fluorescently tagged dyes in the primers or by silver staining the gel prior to scanning' %lthough it is cost effective and can be rather high throughput, silver staining kits for 53,s are being discontinued' 9n addition, many labs are phasing out gels in favor of +2 as the cost of machines becomes more manageable' 3he true po&er of 53,s is in its statistical po&er of discrimination' 9n the ?'5'%', there are 1 loci (#$% locations) that are currently used for discrimination' 7ecause these loci are independently assorted (having a certain number of repeats at one locus doesnJt change the likelihood of having any number of repeats at any other locus), the po&er rule of statistics can be applied' 3his means that if someone has the #$% type of %7+, &here the three loci &ere independent, &e can say that the probability of having that #$% type is the probability of having type % times the probability of having type 7 times the probability of having type +' 3his has resulted in the ability to generate match probabilities of in a quintillion ( &ith 0 "eros after it) or more'

[edit] 8(Chromosome +nalysis

,ecent innovations have included the creation of primers targeting polymorphic regions on the TBchromosome (TB53,), &hich allo&s resolution of multiple male profiles, or cases in &hich a differential e)traction is not possible' TBchromosomes are paternally inherited, so TB53, analysis can help in the identification of paternally related males' TB 53, analysis &as performed in the 5ally 4emings controversy to determine if 3homas Jefferson had sired a son &ith one of his slaves'

[edit] /itochondrial analysis

For highly degraded samples, it is sometimes impossible to get a complete profile of the 1 +F#95 53,s' 9n these situations, mitochondrial #$% (mt#$%) is sometimes typed due to there being many copies of mt#$% in a cell, &hile there may only be B: copies of the nuclear #$%' Forensic scientists amplify the 4O and 4O: regions of the mt#$%, then sequence each region and compare single nucleotide differences to a reference' 7ecause mt#$% is maternally inherited, directly linked maternal relatives can be used as match references, such as oneJs maternal grandmotherJs sisterJs son' % difference of t&o more nucleotides is generally considered to be an e)clusion' 4eteroplasmy and polyB+ differences may thro& off straight sequence comparisons, so

some e)pertise on the part of the analyst is required' mt#$% is useful in determining unclear identities, such as those of missing persons &hen a maternally linked relative can be found' mt#$% testing &as used in determining that %nna %nderson &as not the ,ussian princess she had claimed to be, %nastasia ,omanov' mt#$% can be obtained from such material as hair shafts and old bonesNteeth'

[edit] Considerations hen e)aluating D"+ e)idence

9n the early days of the use of genetic fingerprinting as criminal evidence, Auries &ere often s&ayed by spurious statistical arguments by defense la&yers along these lines: given a match that had a in < million probability of occurring by chance, the la&yer &ould argue that this meant that in a country of say =8 million people there &ere : people &ho &ould also match the profile' 3his &as then translated to a in : chance of the suspect being the guilty one' 3his argument is not sound unless the suspect &as dra&n at random from the population of the country' 9n fact, a Aury should consider ho& likely it is that an individual matching the genetic profile &ould also have been a suspect in the case for other reasons' %nother spurious statistical argument is based on the false assumption that a in < million probability of a match automatically translates into a in < million probability of innocence and is kno&n as the prosecutorJs fallacy' When using ,F(*, the theoretical risk of a coincidental match is in 88 billion ( 88,888,888,888)' 4o&ever, the rate of laboratory error is almost certainly higher than this, and often actual laboratory procedures do not reflect the theory under &hich the coincidence probabilities &ere computed' For e)ample, the coincidence probabilities may be calculated based on the probabilities that markers in t&o samples have bands in recisel# the same location, but a laboratory &orker may conclude that similar BB but not precisely identical BB band patterns result from identical genetic samples &ith some imperfection in the agarose gel' 4o&ever, in this case, the laboratory &orker increases the coincidence risk by e)panding the criteria for declaring a match' ,ecent studies have quoted relatively high error rates &hich may be cause for concern [ !' 7ecause of this, arbitrary ceilings &ere put on match probabilities used in ,F(* analysis rather than the theoretically computed ones' 3oday, ,F(* has become &idely disused due to these difficulties in interpretation' 53,s do not suffer from such subAectivity and provide much better po&ers of discrimination, for unrelated individuals (of the order of in 8R:/ if using a full profile) 9t should be noted that figures of this magnitude are not considered to be statistically suportable by scientists in the ?-, for unrelated individuals &ith full matching #$% profiles a match probability of in a billion (one thousand million) is considered statistically supportable (5ince //0 the #$% profiling system supported by 3he $ational #$% #atabase in the ?- is the 5@.Y #$% profiling system &hich includes 8 53, regions and a se) indicating test, this test updated the 5@. #$% profiling system on &hich the $ational #$% #atabase &as founded in //<' 3he 5@. system included = out of the 8 53, regions used in the 5@.Y system and the same se) indicating test, ho&ever the discriminating po&er of the 5@. system &as only

considered to be supportable at in a million) ' 4o&ever, &ith any #$% technique, the cautious Auror should not convict on genetic fingerprint evidence alone if other factors raise doubt' +ontamination &ith other evidence (secondary transfer) is a key source of incorrect #$% profiles and raising doubts as to &hether a sample has been adulterated is a favorite defense technique' .ore rarely, +himerism is one such instance &here the lack of a genetic match may unfairly e)clude a suspect' When evaluating a #$% match, the follo&ing questions should be asked:

+ould it be an accidental random matchZ 9f not, could the #$% sample have been plantedZ 9f not, did the accused leave the #$% sample at the e)act time of the crimeZ 9f yes, does that mean that the accused is guilty of the crimeZ

[edit] Fa6e D"+ e)idence

3he value of #$% evidence has to be seen in light of recent cases &here criminals planted fake #$% samples at crime scenes' 9n one case, a criminal even planted fake #$% evidence in his o&n body: #r' John 5chneeberger of +anada raped one of his sedated patients in //: and left semen on her under&ear' *olice dre& 5chneebergerJs blood and compared its #$% against the crime scene semen #$% on three occasions, never sho&ing a match' 9t turned out that he had surgically inserted a *enrose drain into his arm and filled it &ith foreign blood and anticoagulants'

[edit] Cases
9n /0>, 7ritish baker +olin *itchfork &as the first criminal caught using #$% fingerprinting' 9n /0>, Florida rapist 3ommie (ee %ndre&s &as the first person to be convicted as a result of #$% evidence, for raping a &oman during a burglaryK he &as convicted on = $ovember /0> and sentenced to :: years in prison'[:![1! 9n /0/, +hicago man @ary #otson &as the first person &hose conviction &as overturned using #$% evidence' 9n // , %llan (egere &as the first +anadian to be convicted as a result of #$% evidence, for four murders he had committed &hile an escaped prisoner in /0/' #uring his trial, his defense argued that the relatively shallo& gene pool of the region could lead to false positives' 9n //:, #$% evidence &as used to prove that $a"i doctor Josef .engele &as buried in 7ra"il under the name Wolfgang @erhard'

3he science &as made famous in the ?nited 5tates in //; &hen prosecutors heavily relied on E and through e)pert &itnesses e)haustively presented and e)plained E #$% evidence allegedly linking F'J' 5impson to a double murder' 3he case also brought to light the laboratory difficulties and handling procedure mishaps &hich can cause such evidence to be significantly doubted' 9n //;, ,+.* detectives successfully tested hairs from a cat kno&n as 5no&ball, and used the test to link a man to the murder of his &ife, thus marking for the first time in forensic history the use of nonBhuman #$% to identify a criminal' 9n //0, #r' ,ichard J' 5chmidt &as convicted of attempted secondBdegree murder &hen it &as sho&n that there &as a link bet&een the viral #$% of the human immunodeficiency virus (49O) he had been accused of inAecting in his girlfriend and viral #$% from one of his patients &ith fullBblo&n %9#5' 3his &as the first time viral #$% fingerprinting had been used as evidence in a criminal trial' 9n :88:, #$% testing &as used to e)onerate #ouglas 2chols, a man &ho &as &rongfully convicted in a /0= rape case' 2chols &as the ;th person to be e)onerated through postBconviction #$% testing' 9n :881, Welshman Jeffrey @afoor &as convicted of the /00 murder of (ynette White, &hen crime scene evidence collected : years earlier &as reBe)amined using 53, techniques, resulting in a match &ith his nephe&'[;! 3his may be the first kno&n e)ample of the #$% of an innocent yet related individual being used to identify the actual criminal, via 6familial searching6' 9n June of :881, because of ne& #$% evidence, #ennis 4alstead, John -ogut and John ,estivo &on a reBtrial on their murder conviction' 3he three men had already served eighteen years of their thirty plus year sentences' 3he trial of ,obert *ickton is notable in that #$% evidence is being used primarily to identify the victims, and in many cases to prove their e)istence' 9n .arch :881, Josiah 5utton &as released from prison after serving four years of a t&elve year sentence for a se)ual assault charge' Questionable #$% samples taken from 5utton &ere retested in the &ake of the 4ouston *olice #epartmentJs crime lab scandal of mishandling #$% evidence' 9n #ecember :88<, ,obert +lark &as proven innocent of a /0 attack on an %tlanta &oman after serving t&enty four years in prison' .r +lark is the =;th person in ?nited 5tates and the fifth in @eorgia to be freed using postBconviction #$% testing'

[edit] !ee also

$ational #$% #atabase restriction fragment length polymorphism (,F(*)

paternity test genealogical #$% test short tandem repeat (53,) capillary electrophoresis (+2) @ene mapping 8arve# v. 8oran

D"+ computing
From Wikipedia, the free encyclopedia

Jump to: navigation, search D"+ computing is a form of computing &hich uses #$% and molecular biology, instead of the traditional siliconBbased computer technologies' 3his field &as initially developed by (eonard %dleman of the ?niversity of 5outhern +alifornia' 9n //;, %dleman demonstrated a proofBofBconcept use of #$% as a form of computation &hich &as used to solve the sevenBpoint 4amiltonian path problem' 5ince the initial %dleman e)periments, advances have been made, and various 3uring machines have been proven to be constructable' 3here are &orks over one dimensional lengths, bidimensional tiles, and even three dimensional #$% graphs processing' Fn %pril :0, :88;, 2hud 5hapiro, Taakov 7enenson, 7inyamin @il, ?ri 7enB#or, and ,ivka %dar at the Wei"mann 9nstitute announced in the Aournal $ature that they had constructed a #$% computer' 3his &as coupled &ith an input and output module and is capable of diagnosing cancerous activity &ithin a cell, and then releasing an antiBcancer drug upon diagnosis' #$% computing is fundamentally similar to parallel computing in that it takes advantage of the many different molecules of #$% to try many different possibilities at once' For certain speciali"ed problems, #$% computers are faster and smaller than any other computer built so far' 7ut #$% computing does not provide any ne& capabilities from the standpoint of computational comple)ity theory, the study of &hich computational problems are difficult to solve' For e)ample, problems &hich gro& e)ponentially &ith the si"e of the problem (2U*5*%+2 problems) on von $eumann machines still gro& e)ponentially &ith the si"e of the problem on #$% machines' For very large 2U*5*%+2 problems, the amount of #$% required is too large to be practical' (Quantum computing, on the other hand, does provide some interesting ne& capabilities)' #$% computing overlaps &ith, but is distinct from, #$% $anotechnology' 3he latter uses the specificity of WatsonB+rick basepairing to make novel structures out of #$%'

3hese structures can be used for #$% computing, but they do not have to be' %dditionally, #$% computing can be done &ithout using the types of molecules made possible by #$% $anotechnology (as the above e)amples sho&)'

[edit] !ee also

*eptide computing *arallel computing

[edit] References

(eonard .' %dleman (:88;' B ). ".olecular +omputation Ff 5olutions 3o +ombinatorial *roblems". -cience (9ournal) 266 (11)2 10/1:10/;. E 3he first #$% computing paper' #escribes a solution for the directed 4amiltonian path problem' .artyn %mos (June :88<)' !heoretical and E$ erimental DNA Com utation' 5pringer' 957$ 1B<;8B=<>>1B0. E 3he first general te)t to cover the &hole field' #an 7oneh, Christo her Dun)orth, Richard <. 3i ton, and <iri -gall (1446). "Fn the +omputational *o&er of #$%". DA,A!82 Discrete A lied ,athematics and Combinatorial = erations Research and Com uter -cience 71. E #escribes a solution for the boolean satisfiability problem' @heorge *aun, @r"egor" ,o"enberg, %rto 5alomaa (Fctober //0)' DNA Com uting ' Ne) Com uting Paradigms' 5pringerBOerlag' 957$ 1B<;8B=; /=B1. E 3he book starts &ith an introduction to #$%Brelated matters, the basics of biochemistry and language and computation theory, and progresses to the advanced mathematical theory of #$% computing' (ila -ari, @reg @loor, 5heng Tu (January :888)' 6?sing #$% to solve the 7ounded *ost +orrespondence *roblem". !heoretical Com uter -cience 231 (/)2 14/:/0>. E #escribes a solution for the bounded *ost correspondence problem, a hardBonBaverage $*Bcomplete problem' 3he history of the 9nternational .eeting on #$% +omputing (*roceedings B (inks) BB [ !