DNA Typing RFLP & PCR-based Systems

Slight but unique differences in the banding pattern of DNA fragments from different individuals of a species are observed when subjected to restriction enzyme analysis. These variations in the DNA are called restriction fragment length polymorphisms !"#$s% and are used as mar&ers on both physical and genetic lin&age maps. Such differences in the !"#$ profiles have revolutionized criminal investigations and have become powerful tools in the identification of individuals in paternity and maternity cases' population genetics' and in the diagnosis of a variety of diseases. (ore recently' polymerase chain reaction $)!%*based tests have been used to obtain a DNA profile. Such tests are generally less involved' providing rapid results that can serve as an alternative or as a complement to !"#$ tests. This integrated laboratory and lecture course is designed to provide the participant with a basic understanding of the principles and methodologies used in !"#$ and $)!*based analyses. The practical applications of !"#$ analysis' coupled with the use of $)!*amplified products the +NT! locus D,S-.' are emphasized in selected topics for the forensic and research scientist as well as for those in the legal community. )ontinuing /ducation 0nits )/0s% available.

Topics Understanding RFLP, VNTR, and Micr sate!!ite "rgani#ati n P$ri%icati n and Preparati n % DNA % r RFLP Ana!ysis &e! '!ectr p( resis and )! tting *ybridi#ati n Pr ced$res PCR Fingerprinting % t(e VNTR D+S,- L c$s N n-.s t pic Pr be Synt(esis and Detecti n by C(emi!$minescence Rand m Amp!i%ied P !ym rp(ic DNA Ana!ysis

Two different DNA tests exist, the RFLP and the PCR. Both are very accurate, but they re conducted in different ways. The restriction fra!"ent #en!th $o#y"or$his" or RFLP %so"eti"es ca##ed DNA fin!er$rintin! or $rofi#in!& is considered to be the "ore accurate of the two. This test exa"ined se'uences of base $airs in a section of a DNA strand with a hi!h $robabi#ity of bein! entire#y uni'ue to the donor. (hen a "atch is found, there is no 'uestion that the donor was at the scene of the cri"e. )t s very conc#usive and fina#i*in!. +nfortunate#y, the RFLP re'uires "any sa"$#e ce##s fro" the cri"e scene #i,e severa# strands of hair or #ar!e s$#atters of b#ood. The ce##s have to be -fresh., too//that is, unda"a!ed and recent#y dead. The test ta,es anywhere fro" 0 wee,s to three "onths to co"$#ete, a#so.

A#thou!h the PCR test, or $o#y"erase chain reaction test, isn t 'uite as accurate, it ta,es "uch #ess ti"e to co"$#ete//a wee, at "ost. The test can be $erfor"ed with "inute cri"e scene sa"$#es, to, which he#$s investi!ators who have #itt#e $hysica# evidence. The DNA doesn t have to be recent#y co##ected, either1 the PCR test can sti## be $erfor"ed even years after//decades after//the fact, and sti## be 2ust as accurate. This is because the PCT "ethod invo#ves co$yin! the avai#ab#e DNA and ana#y*in! on#y one s$ecific !ene, oftenti"es the !ene ca##ed 3LA D4 a#$ha. 5eneticists #oo, for certain ty$es of the !ene %a##e#es& to deter"ine "atches. )f no "atch is found, the donor was not at the cri"e scene. 6atches are not conc#usive, thou!h, because "any $eo$#e "ay have//and are "ore than #i,e#y to have//the sa"e a##e#es as the donor. 7ti##, 2uries have convicted sus$ects based on PCR test resu#ts. 8ven thou!h DNA is so revo#utionary and has aided in the $rosecution of count#ess cri"ina#s, it hasn t !one unnoticed by critics. )n fact, it s because of its hi!h success rate that "any defense attorneys attac, it whenever the test resu#ts are unfavorab#e to their cases. 9thers be#ieve that DNA tests don t account for differences in ethnicity1 whereas one ethnic !rou$ "ay have a hi!her $robabi#ity of se'uence "atches than another !rou$// in short, not "uch is ,nown about how DNA is se'uenced a"on!st the different races. Peo$#e are a#so concerned about incorrect hand#in! of evidence by $o#ice1 a $ri"e exa"$#e of this was seen in the 9.:. 7i"$son cri"ina# tria#. )f the evidence to be !enetica##y tested isn t hand#ed with extre"e care, the DNA cou#d be da"a!ed, $roducin! fau#ty test resu#ts. 7ti##, thou!h, DNA tests are undeniab#y the best forensic evidence avai#ab#e.

This high performance Bradford reagent exhibits substantially increased linearity of response, and only half the expected protein-to-protein variation of other commercial Bradford protein assays.

Highlights: Detects protein concentrations from 1-1,500 g/ml Ready-to-use dye-binding reagent formulation Fast (almost immediate) color de elopment! read at 5"5 nm #ompatible $it% reducing sugars, reducing substances and t%iols (&able 1') Refrigerated reagent is stable for ( years )uperior linear response o er t%e range of 1(5-1,500 g/ml *daptable to microplates +icro protocol useful for protein concentrations from 1-(5 g/ml

How it Wor s: ,se of #oomassie --(50 Dye in a colorimetric reagent for t%e detection and .uantitation of total protein $as first described by Dr' +arion /radford in 1"011' 2n t%e acidic en ironment of t%e reagent, protein binds to t%e coomassie dye' &%is results in a spectral s%ift from t%e reddis%/bro$n form of t%e dye (absorbance ma3imum at 415 nm) to t%e blue form of t%e dye (absorbance ma3imum at 110 nm) (Figure 1)' &%e difference bet$een t%e t$o forms of t%e dye is greatest at 5"5 nm, so t%at is t%e optimal $a elengt% to measure t%e blue color from t%e coomassie dye-protein comple3' 2f desired, t%e blue color can be measured at any $a elengt% bet$een 505 nm and 115 nm' *t t%e t$o e3tremes (505 nm and 115 nm) t%ere is a loss of about 105 in t%e measured amount of color (absorbance) compared to t%at obtained at 5"5 nm'

!igure ". Reaction sc%ematic for t%e #oomassie 6lus 7 &%e /etter /radford *ssay De elopment of color in coomassie dye-based (/radford) protein assays %as been associated $it% t%e presence of certain basic amino acids (primarily arginine, lysine and %istidine) in t%e protein' 8an der 9aals forces and %ydrop%obic interactions also participate in t%e binding of t%e dye by protein' &%e number of #oomassie dye ligands bound to eac% protein molecule is appro3imately proportional to t%e number of positi e c%arges found on t%e protein' Free amino acids, peptides and lo$ molecular $eig%t proteins do not produce color $it% coomassie dye reagents' 2n general, t%e mass of a peptide or protein must be at least :,000 daltons to be assayed $it% t%is reagent' &%e assay is performed at room temperature and no special e.uipment is re.uired' )imply add t%e sample to t%e tube containing reagent and t%e resultant blue color is measured at 5"5 nm follo$ing a s%ort room-temperature incubation' &%e coomassie dye containing protein assay is compatible $it% most salts, sol ents, buffers, t%iols, reducing substances and metal c%elating agents encountered in protein samples'

!igure #. #oomassie 6lus 7 &%e /etter /radford *ssay 6rotocol' &%e main disad antage of coomassie-based (/radford) protein assay is its incompatibility $it% surfactants at concentrations routinely used to solubili;e membrane proteins' 2n general, t%e presence of a surfactant in t%e sample, e en at lo$ concentrations, causes precipitation of t%e reagent' )ince t%e coomassie dye reagent is %ig%ly acidic, a small number of proteins cannot be assayed $it% t%is reagent due to t%eir poor solubility in t%e acidic reagent' *lso, coomassie reagents result in about t$ice as muc% protein<protein ariation as copper c%elation based assay reagents' 2n addition, coomassie dye stains t%e glass or .uart; cu ettes used to %old t%e solution in t%e spectrop%otometer $%ile t%e color intensity is being measured' (#u ettes can be cleaned $it% strong detergent solutions and/or met%anol $as%es, but use of disposable polystyrene cu ettes eliminates t%e need to clean cu ettes')

!igure $. &ypical color response cur es for /)* and /-- using t%e #oomassie 6lus 7 &%e /etter /radford *ssay Reagent' coomassie-based (/radford) protein assays s%are a number of c%aracteristics' &%e #oomassie (/radford) 6rotein *ssay produces a nonlinear standard cur e' &%e #oomassie 6lus 7 &%e /etter /radford *ssay %as t%e uni.ue ad antage of producing a linear standard cur e o er part of its total $or=ing range' 9%en using bo ine serum albumin (/)*) as t%e standard, t%e #oomassie 6lus 7 &%e /etter /radford *ssay is linear from 1(5 to 1,000 g/ml' 9%en using bo ine gamma

globulin (/--) as t%e standard, t%e #oomassie 6lus 7 &%e /etter /radford *ssay is linear from 1(5 to 1,500 g/ml' &%e complete $or=ing range of t%e #oomassie 6lus 7 &%e /etter /radford *ssay co ers t%e concentration range from 1(5 to 1,000 g/ml for t%e tube protocol and from 1 to (5 g/ml for t%e micro protocol' Table ". Reagents compatible $it% #oomassie 6lus 7 &%e /etter /radford *ssay >it using t%e standard protocol' 2nterferences may be obser ed at t%e stated concentration $%en using t%e +icro *ssay 6rocedure' A""oniu" 7u#fate A*ide NaC# Bri2/0> Bri2/>B C3AP7 7D7 Citrate 8DTA 5#ycine Triton D/;;C 5uanidine 3C# Tween/B< E7CN ;.< 6 <.>? >.< 6 <.<@A? <.<;@? >.<? <.<;@? A<< "6 <.; 6 <.; 6 <.<@A? 0.> 6 <.<;@? 0.< 6 =/6erca$toethano# 687 Na93 Bri2/>@ NP/C< C3AP79 7ucrose Tris 5#ucose Triton D/;<< Triton D/C<> Tween/A< 3C# +rea ;.< 6 ;<< "6 <.; 6 <.<0? <.>? >.<? ;<.<? A.< 6 ;.< 6 <.<@A? <.A>? <.<0;? <.; 6 0.< 6

&%e ready-to-use li.uid #oomassie dye reagents must be mi3ed gently by in ersion ?ust before use' &%e dye in t%ese li.uid reagents spontaneously forms loose aggregates upon standing' &%ese aggregates may become isible after t%e reagent %as been standing for as little as 10 minutes' -entle mi3ing of t%e reagent by in ersion of t%e bottle $ill uniformly disperse t%e dye' *fter binding to protein, t%e dye also forms protein-dye aggregates' Fortunately, t%ese proteindye aggregates can be dispersed easily by mi3ing t%e reaction tube' &%is is common to all li.uid #oomassie dye reagents' )ince t%ese aggregates form relati ely .uic=ly, it is also best to routinely mi3 ( orte3 for (-: seconds) eac% tube or plate ?ust before measuring t%e color' %eferences: 1' (' :' 4' /radford, +' (1"01)' Anal. Biochem. &#, (4@-(54' -lo er, /'6' and +cAenry, #')' ((001)' Cell "'(, "(5-":4' >agan, *', et. al. ((000)' J. Biol. Chem. #&(, 11(41-11(4@' -oel, R', et al' ((00()' J. Biol. Chem. #&&, 1@140-1@14@'

%elated )roducts: #oomassie (/radford) 6rotein *ssay >it #oomassie Dry 6rotein *ssay 6lates #ompat-*ble 6rotein *ssay >its /#* 6rotein *ssay >it /#* 6rotein *ssay - Reducing *gent #ompatible +icro /#* 6rotein *ssay >it /)* B /-- 6rotein *ssay )tandard )ets

+odified Co$ry 6rotein *ssay >it Fluoralde%yde Reagent %elated *in s: 6rotein<protein ariation bet$een protein assays #oomassie 6lus 6rotein *ssay Reagent Fre.uently *s=ed Duestions 6ierce 6rotein *ssay &ec%nical Aandboo= Duantitate immobili;ed protein (6DF) E3tinction coefficients guide (6DF) Ao$ to use a protein assay standard cur e (6DF) Determine acceptable $a elengt%s for measuring protein assays (6DF) +ompatible ,nstrumentation: /io+ate : ,8-8is )pectrop%otometer /io+ate 5 ,8-8is )pectrop%otometer FanoDrop 1000 ,8/8is )pectrop%otometer FanoDrop @000 ,8/8is @ )ample )pectrop%otometer

9rderin! )nfor"ation Buy Product Description #

Certificate of Ana#ysis Pkg. Files Price Size Eit

)nstruction Boo, with Protoco#s 67D7

Loca# A0A0@ contact

Coo"assie P#us / The Better Bradford Assay Eit

Sufficient reagents to perform 630 standard assays or 3,160 microplate assays. >it contains< #oomassie 6lus - &%e /etter /radford *ssay "50 ml Reagent *lbumin )tandard (( 10 3 1 ml mg/ml) ampules

Perbio Science N.V. )ndustrie*one ))) )ndustrie#aan AF B/G0A< 8re"bode!e", Aa#st Te#H I0A >0 B> F; BC FaxH I0A >0 B> FA AB 8"ai#H euro"ar,etin!J$erbio.co" Assistance Techni'ue Te#H I0A >0 B> F; GA FaxH I0A >0 B> FA AB 8"ai#H eurotechJ$erbio.co" (ebH www.$erbio.co"

Loca# A0A0B contact

Coo"assie P#us / The Better Bradford Assay Rea!ent

)ufficient reagents to perform (00 standard assays or 1,000 microplate assays' *lbumin )tandard not included'

0<< 6L

Perbio Science N.V. )ndustrie*one ))) )ndustrie#aan AF B/G0A< 8re"bode!e", Aa#st Te#H I0A >0 B> F; BC FaxH I0A >0 B> FA AB 8"ai#H euro"ar,etin!J$erbio.co"

Assistance Techni'ue Te#H I0A >0 B> F; GA FaxH I0A >0 B> FA AB 8"ai#H eurotechJ$erbio.co" (ebH www.$erbio.co"

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AMDIS Downlo d P ge
The Auto"ated 6ass 7$ectra# Deconvo#ution and )dentification 7yste" %A6D)7& is a co"$uter $ro!ra" that extracts s$ectra for individua# co"$onents in a 5CK67 data fi#e and identifies tar!et co"$ounds by "atchin! these s$ectra a!ainst a reference #ibrary. )t was deve#o$ed at N)7T with su$$ort fro" the +nited 7tates De$art"ent of Defense and is free#y avai#ab#e. Avai#ab#e here is the current re#ease of A6D)7 %Lersion A.@>, February A<<F Re#ease&. 7tartin! fro" version A.@0, A6D)7 uses a new 3e#$ fi#e for"at1 the C36 or co"$i#ed ht"# he#$ "ode#. Qou can a#so down#oad it se$arate#y be#ow and exa"ine it inde$endent#y of A6D)7. The 3e#$ fi#e is a#so $rovided se$arate#y in PDF for"at1 which can a#so be down#oaded se$arate#y be#ow. As a new user to A6D)7 you are ur!ed to read Cha$ters ; and A of the he#$ in order to understand the basic features of A6D)7. 8ven if you ,now A6D)7 a#ready, you wi## find the tutoria# sty#e a$$roach of Cha$ter A to be infor"ative and very easy to fo##ow.

Changes in AMDIS
This re#ease of A6D)7 %Lersion A.@>& has a nu"ber of "inor i"$rove"ents co"$ared to version A.@C1 $ri"ari#y these have been re'uested by the users. 7o"e bu!s have a#so been fixed. The i"$rove"ents are as fo##owsH

;. 6u#ti$#e 7can Ran!es / A6D)7 now su$$orts "u#ti$#e scan ran!es / that is different "K* scan ran!es at different ti"es in the chro"ato!ra". +se Ana#y*eK7ettin!sK7can 7ets and $ut the 7tart and 8nd ti"e as we## as the Low and 3i!h "K* for each scan set and then state the Nu"ber of 7ets. A#so use Ana#y*eK7ettin!sK)nstr and se#ect +se 7can 7ets. For "ost cases this wi## not have a bi! effect, but it wi## he#$ !et better resu#ts for so"e cases where the scan ran!e chan!es a #ot fro" the be!innin! of the 5C run to the end. A. Co"$onent (idth / The Co"$onent (idth can now be a "axi"u" of 0A scans on either side of the co"$onent "axi"u" rather than A< %set usin! Ana#y*eK7ettin!sKDeconv&. 3owever, it is not !ood chro"ato!ra$hic $ractice to have a hi!h nu"ber of scans across an iso#ated $ea,. )f the ;<? $oints on either side have B scans between the", this is sufficient for !ood 'uantitative wor, and the si!na#/to/noise wi## be better with fewer scan across a $ea,. There are so"e cases where a s$ecific co"$onent is very wide and increasin! the co"$onent width "ay he#$ in its ana#ysis. 3owever, it is !enera##y better to first try reducin! the scan s$eed used for ac'uisition to "a,e the $ea, narrower1 since "ore is not a#ways better. 0. Retention )ndex Data / )f you want to have retention index data ca#cu#ated, but not used, set the Ty$e of Ana#ysis e'ua# to +se Retention )ndex Data and the 6axi"u" Pena#ty to < %set usin! Ana#y*eK7ettin!sKDeconv&. The for"er, so"ewhat confusin! "ethod of !ettin! this sa"e resu#t has been o"itted. C. 6atch Factor Pena#ty / There is now a Ssudden deathS cut off for the 6atch Factor Pena#ty %set usin! Ana#y*eK7ettin!sK)dentif&. The #eve#s are now (ea,, Avera!e, 7tron!, Lery 7tron! and )nfinite and these corres$ond, res$ective#y, to a ;, >, ;<, A< and ;<<< "atch factor $oint $ena#ty $er R) window. For exa"$#e, if you set the R) (indow to ;< and the observed R) differs #ess than ;< %ie, ; window& fro" the #ibraryTs R) va#ue, there wi## be no $ena#ty a$$#ied to the "atch factor. 3owever, when the difference is !reater than ;<, there wi## a $ena#ty of ;, >, ;<, A< or ;<<< $er R) (indow %ie, ;<& beyond the ;<. 7o for exa"$#e, if the observed R) is ;@ different fro" the #ibraryTs R) va#ue, there wi## be a @K;< %N<.@& $ena#ty a$$#ied1 and so <.@, 0, @, ;A or @<< $oints. 7ince you start with ;<< as a $erfect "atch factor va#ue, you can see that the )nfinite settin! wi## act as a very 'uic, cut off. >. 7e#ection of Best 6atch Factor / There have been so"e a#!orith"ic chan!es in how A6D)7 se#ects the best "atch factor if you are not usin! 6u#ti$#e )dentifications $er Co"$ound %contro##ed usin! Ana#y*eK7ettin!sK)dentif&. Now if there are two or "ore "atch va#ues that are #ar!e %UG>&, A6D)7 wi## se#ect the one that has the "axi"u" area. This wi## "ore often identify the co"$onent that you thin, is the best. @. 8xtra (idth / A6D)7 has a#ways used "u#ti$#e a#!orith"s for areas and co"$utes two va#ues ca##ed )nt!r. 7i!na# %inte!rated si!na#& and Area. The )nt!r. 7i!na# va#ue is the area under the co"$onent1 where the #atter can be seen !ra$hica##y by ri!ht/c#ic,in! in the u$$er T)C window and chec,in! the 7how Co"$onent on Chro"ato!ra" "enu ite". 3owever, there are a#ternative ways to assess a base #ine and these wi## so"eti"es resu#t in the co"$onentTs base#ine bein! extended by one or "ore scans to one or both sides of the ori!ina# base#ine.

This a#ternative area assess"ent is re$orted as the Area va#ue and wi## be the sa"e as )nt!r. 7i!na#, if no extension was deter"ined, or otherwise wi## be #ar!er. Previous#y there was no infor"ation re$orted about this a#ternative base#ine, but now it is re$orted as 8xtra (idth %in the Co"$onent section of the )nfor"ationa# Lists window&. )ts for"at is a/b1 where, for exa"$#e, </A wou#d "ean that the base#ine was extended by no scans to the #eft and two scans to the ri!ht of the ori!ina# base#ine. )f you want to bui#d a ca#ibration tab#e %externa# to A6D)7&, you can use either one or the other area ty$es, but do not "ix the". (e do not thin, that there is "uch difference between the two, but you "ay find one "ay be better for so"e ty$es of ana#ysis. F. RT Ana#ysis / Qou can now do an A6D)7 ana#ysis usin! #ibrary RT va#ues as an a#ternative to R) va#ues %set Ty$e of Ana#ysis N +se Retention Ti"e&. 3owever, we sti## reco""end the use of R) since there are extensive #iterature va#ues for R) that can he#$ you. B. R)KRT (indow and 6atch Factor Pena#ties / The R)KRT (indow and 6atch Factor Pena#ties $ara"eters %set usin! Ana#y*eK7ettin!sK)dentif& are now auto"atica##y se#ectedKc#eared accordin! to the Ty$e of Ana#ysis se#ected. NoteH 7u$$ort for different co#u"n ty$es in the Co"$ound 8ditor used for a Tar!et Co"$ounds Library %67L& has been re"oved. The reference to co#u"n ty$e se#ection %$revious#y set usin! Ana#y*eK7ettin!sK)dentif& has corres$ondin!#y been re"oved. As a#ways, we are interested in your co""ents and su!!estions as to how we can "a,e A6D)7 a better too# for your use.

-ownload .ection:
There are few ty$es of down#oads / se#ect the ones that you need. Description !" bit #ersion Size Fu## Pro!ra" )nsta##ationH Pro!ra" fi#es, 3e#$, 7a"$#e Data Down#oad V;;.;6B 3e#$ fi#e %W.C36 for"at& Down#oad V0.F6B 6anua# %W.PDF for"at& Down#oad VC.C6B 7a"$#e Data Down#oad V0.@6B

Other Versions
The version avai#ab#e at this site has been tested on these 0A bit versions of 67 (indowsH (indows A<<<, (indows NT C.<, (indows G> or (indows GB. A ;@/bit version and a Sb#indedS version %on#y infor"ation concernin! identified co"$ounds is shown& can a#so be down#oaded. Contact the N)7T 6ass 7$ectro"etry Data Center for further infor"ation. Down#oad ;@ bit version

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The first version of A6D)7 was re#eased 7e$te"ber ;GG@ and has been distributed at no additiona# cost a#on! with the N)7T 6ass 7$ectra# Database since :anuary ;GGB. )t was a#so inc#uded as $art of the N)7T 67 Database De"o $ac,a!e.

Searching NIST/EPA/NIH Mass Spectral Library

(hi#e users "ay deve#o$ their own tar!et #ibraries, in order for A6D)7 to search the N)7TK8PAKN)3 6ass 7$ectra# Library, a co$y of the current version of this #ibrary %N)7T <A or #ater& as we## as the N)7T 67 7earch Pro!ra" %version ;.@ or hi!her& is re'uired. N)7T 6ass 7$ectra# Library is avai#ab#e on#y fro" distributors. The N)7T 67 7earch Pro!ra" u$!rades can be down#oaded.

More In or!ation
For a brief ex$#anation of A6D)7 and an overview of the $ro!ra"H A6D)7 8x$#anation A6D)7 9verview The co"$#ete "anua# for A6D)7 is avai#ab#e as a PDF fi#e %#in, to down#oad is above&. )t is a#so avai#ab#e as a 6icrosoft (ord X docu"ent a#on! with additiona# test data for A6D)7. The "anua# and sa"$#e data $ac,a!e are archived in a R)P fi#es, with additiona# instructions in a read"e fi#e. This data is su$$#ied for use with the S4uic, 7tart A6D)7S #ocated in A$$endix A of the A6D)7 3e#$ syste". AlgorithmsH Detai#s of a#!orith"s e"$#oyed in A6D)7 are described in the $a$er SAn )nte!rated 6ethod for 7$ectru" 8xtraction and Co"$ound )dentification fro" 5CK67 DataS which a$$eared in the Journal of the American Society of Mass Spectrometry in Lo#u"e ;<, ;GGG, $a!es FF</FB;. This $a$er is avai#ab#e in PDF for"atH A6D)7 6ethod Pa$er The N)7T 67 7earch Pro!ra" v A.< can be down#oaded se$arate#y.

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