03 Fundamentos de Pcr

8/12/2019 03 Fundamentos de Pcr

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PCR


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Initiation - Forming theReplication Eye

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Origin of Replication

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Leading StrandLeading Strand

Laging StrandLaging Strand

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Extension - The Replication Fork 5

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Single strandbindingproteins

DNAPolymerase

Okazakifragment

RNAPrimers

Primase

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Heli ase


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Functions And TheirAssociated En ymes

!igase"oining nicks

#$A PolymerasePolymeri ing #$A PrimasePro%iding primer

!nzyme"#n tion &elicase ''( Proteins Topisomerase

)elting #$A


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Theoretical *ield Of PCR $%eoreti al yield & ' n x y

+here y , the startingnum er of copies and

n , the num er of thermal cycles

& ()*+3*,+(-'+,))

.f yo# start /it% ()) opies+ %o/ many opies aremade in 3) y les0

' n 1 y

& '3)

1 ())& (+)*3+*,(+-', 1 ())


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&o. The Functions Of ReplicationAre Achie%ed #uring PCR

$/A as fragmentsare short

"oining nicks

Taq #$APolymerase

Polymeri ing #$A

Primers areadded to thereaction mix

Pro%iding primer

P2R "#n tion &eat)elting #$A


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Polymerase Chain Reaction

0in%ented y 1ary )ullis .hile cruising in a&onda Ci%ic on &igh.ay 234 from 'an

Francisco to )endocino5

6It .as 7uiet and something 8ust .ent5 Click96


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)ullis

::: ;PCR is a chemical procedure that .ill makethe structures of the molecules of our genes aseasy to see as ill oards in the desert and aseasy to manipulate as Tinkertoys


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)aking #$A= Components

#$A template5

d$TPs5

#$A Polymerase5

Primer5

En%ironment:


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#$A Template

0a single stranded #$A molecule that

specifies the synthesis of a complementarynucleotide se7uence5

||||||||||||||||||||||||||||||||||||3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC-5


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#eoxynucleotidesd$TPs

0dATP5 dCTP5 d>TP5 and dTTP5

? A5 C5 >5 and T5

? d$TPs:


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#$A Polymerase

0the en yme that cataly es the formation of#$A from deoxynucleotides @d$TPs 5


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Oligonucleotides

::::short pieces of synthetic #$A can emanufactured that contain any se7uence:


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Primer

0dou le stranded #$A is re7uired for theinitiation of synthesis y polymerases5

0a short single stranded #$A is necessaryfor the functioning of #$A polymerases iftemplates are single stranded5

||||||||||||||||||||||||||||||||||||3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5

GCATGCATTAG

oligo primer


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Primer Orientation


5--GCATGCATTAG--3

0primers are synthesi ed B -to- 5

0primers are complementary to the strand thatis to e replicated:


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)aking One 'trand Of #$A


5--GCATGCATTAG GCTACATCGACATCGACTAGCACTG--3

Add Polymerase

Add dNTPs


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)aking T.o )ore 'trands



Denature


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#enaturing

0#$A denaturing conditions such as highheat or high salt concentrations irre%ersi ly

denature most polymerases @en yme 5

0d$TPs are not affected y denaturation5

0primers are not affected y denaturation:


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)aking T.o )ore 'trands

3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5


5--GCATGCATTAG

CTGATCGTGAC--5

GCTACATCGACATCGACTAGCACTG--3

3--GCTACGTAATCCGATGTAGCTGTAG

Add polymerase.

add primer to second strand


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#enaturation 'tep (ad

0se%eral rounds of in vitro replication could e performed5 ho.e%er5 accumulation ofdenatured polymerase 7uickly poisons thereaction:


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Thermus aquaticus

0 acteria disco%ered in a hot spring in*ello.stone $atural Park in 2D B5

0li%es in salty .ater that ranges from G o - B o C5

0thus5 does #$A replication at high temperatures:


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Thermus aquaticus En ymes

0 asic research demonstrated that manyen ymes isolated from Thermus aquaticus

function at %ery high temperatures5

0temperatures nearing 2GG o C5

0#$A denaturating temperatures:


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Click

01ary )ullis reali ed that repetiti%e roundsof #$A synthesis could e performed y

using a heat-sta le polymerase5

0 Thermus aquaticus =Taq polymerase:


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94 o


5--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3

5--GCATGCATTAT

CTGATCGTGAC--5

Denature Step~30 seconds

~60 o



5--GCATGCATTAT

CTGATCGTGAC--5

Annealing Step~30 seconds

72 o



5--GCATGCATTAT

CTGATCGTGAC--5


3--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5


3--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5

Synthesis~30 seconds/kb

PCR


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Exponential 'ynthesis

H as fe. as 2 #$A templates re7uired5

H excess d$TP'5

H excess primers5

H multiple cycles:


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PCR Applications0ne. applications are created e%ery day5

H PCR products can e used for mapping genes5

H PCR products can e used as pro es5

H PCR products can e pro ed5

H PCR can e used to identify genotypes5H PCR can e used to se7uence #$A directly:


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7.7 Polymerase chain reaction: analternative to cloning

H The polymerase chain reaction (PCR) can be used toamplify rare specific DNA sequences from a complexmixture when the ends of the sequence are known

H PCR amplification of mutant alleles allows detectionof human genetic diseases

H DNA sequences can be amplified by PCR for use incloning, as probes, and in forensics


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7.7 The polymerase chain reaction

Figure 7-38


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Rea i n en adena de la polimerasa 4 P2R

PCR es bsicamente una tcnica de amplificacindel DNA.

PCR

amplificacin

DNA

(molecule sencilla)

Muchasmoleculas


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H S ntesis por DNA polimerasa

H - A T G C A T G C A T G C * *

A C G- T


Cortos primers de DNA espec ficos hibridi!an con lacadena "ue tiene "ue ser copiada

#$ %$


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A C G T- T


#$ %$


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A C G T- T A #$ %$



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A C G T- T A C


#$ %$


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A C G T- T A C G


#$ %$


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A C G T- T A C G T


#$ %$


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A C G T- T A C G T A


#$ %$


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Despues del primer periodo de sintesis deDNA& tenemos copiada una cadena de DNA

Primer 2 ' tensin de la cadena

Cadena ori inal de DNA

*Cmo produce este proceso una AMP+,-,CAC, N/



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Primero& la fusin separa las dos cadenas de DNA a alta

temperatura (01223C)



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temperatura (01223C)



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temperatura (01223C)+ue o& usando un se undo primer& se copia la nue4acadena con la DNA polimerasa

Al mismo tiempo& la cadena ori inal se copianue4amente& dado "ue ha5 un e ceso de Primer 1

6

1

Ahora tenemos dos copias de la molcula ori inal



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Para controlar el proceso& necesitamos controlar la temperatura

7i repetimos el ciclo& tendremos cuatro copias


-usin8#3C

9ibridi!acin#2:;2


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&ay componentes esenciales en

el proceso standard de PCR H A#$ polimerasa termoesta leH Oligonucleotidos iniciadores @;primers C entre G- GQ@c E%itar las secuencias repetidas


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@c E%itar las secuencias repetidas

#?#?

%?%?

#?#?%?

%?

D mero de primer

PCR%?repetido

#$:NNNNNNNNNNNNN@A@A:%$#$:NNNNNNNNNNNNN@A@A:%$

#$:NNNNNNNN@A@A:%$ %$:A@A@NNNNNNN


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#?#?

%?%?

no ha5 e tensin

PCR#? repetido

#?:@A@ANNNNNNNNNNNNN:%?#?:@A@ANNNNNNNNNNNNN:%?

#?:@A@ANNNNNNNN%?:NNNNNNNNA@A@:#?

#?#?

%?%?


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#?

#?

%?

%?

Productos de PCR no deseados

PCR-ormacin de hor"uillas

#$:NNNNNNNNNNN CA@ C:%$#$:NNNNNNNNNNNNNNNNN:%$

#$:NNNNNNNNNNN CA %$:C @

#?

%?


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Oligonucle tidos iniciadores

@primersH 'e conoce su secuenciaH Es el factor mas importante para la eficiencia y la especificidad del

procesoH #e en estar presentes en exceso @2G 2 , G cycles5 2 kH Re7uieren de un cuidadoso diseNo

H Reglas de diseNo=@a longitud , 24-3B

@ Contenido de > C entre G- GQ@c E%itar las secuencias repetidas@d alores de T m de no mas de BKC uno del otro


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A#$ molde @templadoH Puede ser ss#$A o ds#$A @simple o do le

cadenaH A#$ circular y cerrado es le%emente menos

efecti%o 7ue el A#$ linealH Ssualmente se utili an %arios miles de copias5 e8=

2 g de humano5 2G ng de le%adura5 2 ng de acteriano o 2 pg of plasmJdico

H 'e puede amplificar a partir de una sola molUculade A#$ molde5 pero las condiciones de en estarmuy optimi adas


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El ciclo de PCRH #esnaturali aci n - D -DBKC por B segundos si > C V BBQH Temperatura de hi ridi aci n @Annealing ? de e ser calculada

o determinada empiricamente para cada par de primersdemasiado alta , poco o nada de producto

demasiado a8a , annealing no especJfico , productosincorrectosH Extensi n -

a la temperatura ptima de la A#$ polimerasa utili adae8:= 3KC para Taq

2 min/k de longitud5 dado 7ue la Taq polimeri a 3GGG p /min

solo a partir del r ciclo son producidos A#$ duplex de lalongitud deseada5 los cuales a partir de allJ se tornan en el

producto principal


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El ciclo de PCR H #esnaturali aci n - D -DBKC por B segundos si > C V BBQH Temperatura de hi ridi aci n @Annealing ? de e ser calculada o determinada empiricamente

para cada par de primers

demasiado alta , poco o nada de productodemasiado a8a , annealing no especJfico , productos incorrectos

H Extensi n -a la temperatura ptima de la A#$ polimerasa utili ada

e8:= 3KC para Taq

2 min/k de longitud5 dado 7ue la Taq polimeri a 3GGG p /min

solo a partir del r ciclo son producidos A#$ duplex de la longitud deseada5 los cuales a partir de allJ se tornan en el producto principal

H $Wmero de ciclos -- algunos componentes se tornan limitantes despues de Gciclos @si el nWmero inicial , 2G B molUculas- se necesitan L3B ciclos para amplificar un gen de copiaWnica a partir de A#$ gen mico de mamJfero #$A


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ariantes de la PCR H Touchdo.n PCR H Colony PCR H)ultiplex PCR H&ot start PCR H $ested PCR H In%erse PCR H !ong PCR


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)ultiplex PCR

H #escri e una PCR en la cual hay presentesmultiples pares de primers @hasta 4 lo 7ueda una serie de productos: !os mismos

pueden %erse como mWltiples andas en ungel de agarosa

H )ultiplex PCR es frecuentemente usada en

diagn stico mUdicoH Ahorra templado5 tiempo y gastosH Re7uiere una cuidadosa optimi aci n


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$ested PCR H A %eces 2 ronda de PCR no da un producto Wnico

product a partir de un templado comple8o5apareciendo un ;chorreado #llisDr>?ary @> #llis

HOB DO!S P2R BOR? 0HOB DO!S P2R BOR? 0


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HOB P2R 2A ! A@OC$ :>HOB P2R 2A ! A@OC$ :>


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@ $@ $ CHANCE FAVORS THE PREPARED MIND.CHANCE FAVORS THE PREPARED MIND.

Louis Paster Louis Paster

H $%e dis o6ery of t%e first DNA polymerase

by Art%#r ?ornberg+ (855

H DNA polymerase p#rifi ation+ (85-

H$%ermodynami des ription of DNA annealing andprimer e1tension in t%e 5 to 3 dire tion+ (8554(8*)



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@@

$%e first des ription of P2R t%eory$%e first des ription of P2R t%eory

?leppe+ ?>+ O%ts#ka+ !>+ ?leppe+ R>+ oline#1+ .>+?%orana+ HE >

St#dy on polyn# leotides repair repli ation of s%ortsynt%eti DNAs as atalyzed by DNA polymerases>

F> ol>@iol>+(8*( + 5G 3,(4G(>

HOB P2R 2A ! A@OC$HOB P2R 2A ! A@OC$..


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$%e first P2R e1periment$%e first P2R e1periment Dr> ? ell ?leppe ? ell ?leppe


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@ $@

B%y /as not large4s ale a#tomation of DNAB%y /as not large4s ale a#tomation of DNAamplifi ation p#rs#ed by t%e ?%orana laboratory0amplifi ation p#rs#ed by t%e ?%orana laboratory0

(> $%ey belie6ed t%at t%e goal of in 6itro DNA synt%esis %adbeen a %ie6ed

'> Hig% temperat#re4stable DNA polymerases /ere #nkno/n

3> DNA loning + /%i % %ad #st beg#n+ be ame t%e preferredmet%od for prod# ing large amo#nts of DNA

,> aybe + be a#se of t%e diffi #lty to synt%esize large amo#ntsof primers no one t%o#g%t t%at t%e spe ifi amplifi ation ofgenomi DNA /as feasible


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@@

Dri6ing #p to endo ino and t%inking abo#t an e1periment toDri6ing #p to endo ino and t%inking abo#t an e1periment tolook at one parti #lar letter of t%e geneti ode+ . designed alook at one parti #lar letter of t%e geneti ode+ . designed asystem in my mind> As . repaired t%e t%ings . t%o#g%t o#ld gosystem in my mind> As . repaired t%e t%ings . t%o#g%t o#ld go/rong /it% it+ s#ddenly/rong /it% it+ s#ddenly . generated somet%ing t%at if . did it o6er. generated somet%ing t%at if . did it o6erand o6er again /o#ld be P2R> .t /o#ld go '+ ,+ -+ (G+ 3' > > > in 3)and o6er again /o#ld be P2R> .t /o#ld go '+ ,+ -+ (G+ 3' > > > in 3)y les make as many base pairs from one little region as . %ad iny les make as many base pairs from one little region as . %ad int%e /%ole genomeI $%at /as t%e e#reka point>t%e /%ole genomeI $%at /as t%e e#reka point> . said 9%oly s%itI. said 9%oly s%itI@y p#tting t%e trip%osp%ates JDNA b#ilding blo ksK in t%ere@y p#tting t%e trip%osp%ates JDNA b#ilding blo ksK in t%eremyself+ . o#ld do t%is pro ess o6er and o6er and amplify t%emyself+ . o#ld do t%is pro ess o6er and o6er and amplify t%e

DNA:;DNA:;.nter6ie/ ?ary #llis


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HSaiki R+ ?>M S %arf SM "aloona "M #llis ?> @M Horn E> $M !rli % H>


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A>M Arn%eim N>+!nzymati amplifi ation of beta4globin genomi se7#en es and restri tion site analysis for diagnosis of si kle

Science + (8-5 De ')+ '3) @M "aloona "> A> Spe ifi synt%esis of DNA in 6itro 6ia a polymerase 4 atalyzed %ain rea tion> Methods in Enzymology + (8-* +(55 33545)>

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@

Ta !"A #olymeraseTa !"A #olymerase

H?agan+ S>+ Do%erty+ >+ and Eits%ier+ F>

"e$ England %ournal o& Medicine 3(* 8-5488)>

Saiki R> ?M Eelfand D> HM Stoffel SM S %arf S> FMHig# %i RM Horn E> $M #llis ?> @M !rli % HA>Primer4dire ted enzymati amplifi ation of DNA /it% a

t%ermostable DNA polymerase > Science + (8-- Fan'8+ '38
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@

$%e Nobel Prize in 2%emistry (883

9"or ontrib#tions to t%ede6lopment of met%ods /it%inDNA4based %emistry:;

9"or %is in6ention of polymerase%ain rea tion


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'' Almost overnight( #C) became a standard research Almost overnight( #C) became a standard research

techni ue and practical applications soon &ollo$ed.techni ue and practical applications soon &ollo$ed.

Dete ting pat%ogens #sing genome4spe ifi primer pairs

S reening spe ifi genes for #nkno/n m#tations

Eenotyping #sing kno/n se7#en e tagged sites


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Dete ting pat%ogens #sing genome4spe ifi primer pairs

Appli ations of P2R Appli ations of P2R


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.dentifi ation of geneti m#tations

$%e P2R te %ni7#e of

single strand con&ormational polymorphism *SSC#+sin gle strand con&ormational polymorphism *SSC#+

is one of t%e most /idely #sed met%ods for dete ting single base

pair %anges in genomi DNA]

]

Non4denat#ring a rylamide gelNon4denat#ring a rylamide gel

Appli ations of P2R Appli ations of P2R


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Eenotyping #sing kno/n se7#en e tagged sites


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Lab Appli ations of P2R Lab Appli ations of P2R


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Dire t loning from genomi DNA or DNA

,n vitro m#tagenesis and engineering of DNAHEeneration of spe ifi se7#en es of loned do#ble4stranded DNA for #se as probes

HEeneration of probes spe ifi for #n loned genes bysele ti6e amplifi ation of parti #lar segments of DNA

HEeneration of libraries of DNA from small amo#nts ofmRNA

HEeneration of large amo#nts of DNA for se7#en ing


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irtual PCR ResultsH irtual PCR searches

entire genome lookingfor potential primersites .ithin 2G5GGG

ases of one another:H If found5 it performs a

%irtual PCR reaction:
http://grup.cribi.unipd.it/cgi-bin/mateo/vpcr2.cgihttp://grup.cribi.unipd.it/cgi-bin/mateo/vpcr2.cgi


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Applications of PCR

H )utation testing5 e.g. cystic fi rosis:H #iagnosis or screening of ac7uired diseases5

e.g. AI#':H >enetic profiling in forensic5 legal and io-

di%ersity applications:

H 'ite-directed mutagenesis of genes:H ^uantitation of mR$A in cells or tissues:

Documents

03 Fundamentos de Pcr