Date post: | 03-Jun-2018 |
Category: |
Documents |
Upload: | sol-mln-pizarnik |
View: | 221 times |
Download: | 0 times |
of 99
8/12/2019 03 Fundamentos de Pcr
1/99
PCR
8/12/2019 03 Fundamentos de Pcr
2/99
Initiation - Forming theReplication Eye
3 5
355
5
3
3
Origin of Replication
5
3
3
5
5
3
5
5
5
3
33
8/12/2019 03 Fundamentos de Pcr
3/99
Leading StrandLeading Strand
Laging StrandLaging Strand
3
53
5
Extension - The Replication Fork 5
5
5
3
3
53
3
5
Single strandbindingproteins
DNAPolymerase
Okazakifragment
RNAPrimers
Primase
53
5
Heli ase
8/12/2019 03 Fundamentos de Pcr
4/99
Functions And TheirAssociated En ymes
!igase"oining nicks
#$A PolymerasePolymeri ing #$A PrimasePro%iding primer
!nzyme"#n tion &elicase ''( Proteins Topisomerase
)elting #$A
8/12/2019 03 Fundamentos de Pcr
5/99
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 ())
8/12/2019 03 Fundamentos de Pcr
6/99
&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
8/12/2019 03 Fundamentos de Pcr
7/99
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
8/12/2019 03 Fundamentos de Pcr
8/99
)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
8/12/2019 03 Fundamentos de Pcr
9/99
)aking #$A= Components
#$A template5
d$TPs5
#$A Polymerase5
Primer5
En%ironment:
8/12/2019 03 Fundamentos de Pcr
10/99
#$A Template
0a single stranded #$A molecule that
specifies the synthesis of a complementarynucleotide se7uence5
||||||||||||||||||||||||||||||||||||3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC-5
8/12/2019 03 Fundamentos de Pcr
11/99
#eoxynucleotidesd$TPs
0dATP5 dCTP5 d>TP5 and dTTP5
? A5 C5 >5 and T5
? d$TPs:
8/12/2019 03 Fundamentos de Pcr
12/99
#$A Polymerase
0the en yme that cataly es the formation of#$A from deoxynucleotides @d$TPs 5
8/12/2019 03 Fundamentos de Pcr
13/99
Oligonucleotides
::::short pieces of synthetic #$A can emanufactured that contain any se7uence:
8/12/2019 03 Fundamentos de Pcr
14/99
Primer
0dou le stranded #$A is re7uired for theinitiation of synthesis y polymerases5
0a short single stranded #$A is necessaryfor the functioning of #$A polymerases iftemplates are single stranded5
||||||||||||||||||||||||||||||||||||3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5
GCATGCATTAG
oligo primer
8/12/2019 03 Fundamentos de Pcr
15/99
Primer Orientation
||||||||||||||||||||||||||||||||||||3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5
5--GCATGCATTAG--3
0primers are synthesi ed B -to- 5
0primers are complementary to the strand thatis to e replicated:
8/12/2019 03 Fundamentos de Pcr
16/99
)aking One 'trand Of #$A
||||||||||||||||||||||||||||||||||||3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5
5--GCATGCATTAG GCTACATCGACATCGACTAGCACTG--3
Add Polymerase
Add dNTPs
8/12/2019 03 Fundamentos de Pcr
17/99
)aking T.o )ore 'trands
||||||||||||||||||||||||||||||||||||3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5
5--GCATGCATTAG GCTACATCGACATCGACTAGCACTG--3
Denature
8/12/2019 03 Fundamentos de Pcr
18/99
#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:
8/12/2019 03 Fundamentos de Pcr
19/99
)aking T.o )ore 'trands
3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5
5--GCATGCATTAG GCTACATCGACATCGACTAGCACTG--3
5--GCATGCATTAG
CTGATCGTGAC--5
GCTACATCGACATCGACTAGCACTG--3
3--GCTACGTAATCCGATGTAGCTGTAG
Add polymerase.
add primer to second strand
8/12/2019 03 Fundamentos de Pcr
20/99
#enaturation 'tep (ad
0se%eral rounds of in vitro replication could e performed5 ho.e%er5 accumulation ofdenatured polymerase 7uickly poisons thereaction:
8/12/2019 03 Fundamentos de Pcr
21/99
8/12/2019 03 Fundamentos de Pcr
22/99
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:
8/12/2019 03 Fundamentos de Pcr
23/99
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#$A denaturating temperatures:
8/12/2019 03 Fundamentos de Pcr
24/99
Click
01ary )ullis reali ed that repetiti%e roundsof #$A synthesis could e performed y
using a heat-sta le polymerase5
0 Thermus aquaticus =Taq polymerase:
8/12/2019 03 Fundamentos de Pcr
25/99
94 o
3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5
5--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3
5--GCATGCATTAT
CTGATCGTGAC--5
Denature Step~30 seconds
~60 o
3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5
5--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3
5--GCATGCATTAT
CTGATCGTGAC--5
Annealing Step~30 seconds
72 o
3--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5
5--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3
5--GCATGCATTAT
CTGATCGTGAC--5
5--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3
3--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5
5--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3
3--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5
Synthesis~30 seconds/kb
PCR
8/12/2019 03 Fundamentos de Pcr
26/99
Exponential 'ynthesis
H as fe. as 2 #$A templates re7uired5
H excess d$TP'5
H excess primers5
H multiple cycles:
8/12/2019 03 Fundamentos de Pcr
27/99
8/12/2019 03 Fundamentos de Pcr
28/99
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 #$A directly:
8/12/2019 03 Fundamentos de Pcr
29/99
7.7 Polymerase chain reaction: analternative to cloning
H The polymerase chain reaction (PCR) can be used toamplify rare specific DNA sequences from a complexmixture when the ends of the sequence are known
H PCR amplification of mutant alleles allows detectionof human genetic diseases
H DNA sequences can be amplified by PCR for use incloning, as probes, and in forensics
8/12/2019 03 Fundamentos de Pcr
30/99
7.7 The polymerase chain reaction
Figure 7-38
8/12/2019 03 Fundamentos de Pcr
31/99
Rea i n en adena de la polimerasa 4 P2R
PCR es bsicamente una tcnica de amplificacindel DNA.
PCR
amplificacin
DNA
(molecule sencilla)
Muchasmoleculas
8/12/2019 03 Fundamentos de Pcr
32/99
H S ntesis por DNA polimerasa
H - A T G C A T G C A T G C * *
A C G- T
Rea i n en adena de la polimerasa 4 P2R
Cortos primers de DNA espec ficos hibridi!an con lacadena "ue tiene "ue ser copiada
#$ %$
8/12/2019 03 Fundamentos de Pcr
33/99
H S ntesis por DNA polimerasa
H - A T G C A T G C A T G C * *
A C G T- T
Rea i n en adena de la polimerasa 4 P2R
#$ %$
8/12/2019 03 Fundamentos de Pcr
34/99
H S ntesis por DNA polimerasa
H - A T G C A T G C A T G C * *
A C G T- T A #$ %$
Rea i n en adena de la polimerasa 4 P2R
8/12/2019 03 Fundamentos de Pcr
35/99
H S ntesis por DNA polimerasa
H - A T G C A T G C A T G C * *
A C G T- T A C
Rea i n en adena de la polimerasa 4 P2R
#$ %$
8/12/2019 03 Fundamentos de Pcr
36/99
H S ntesis por DNA polimerasa
H - A T G C A T G C A T G C * *
A C G T- T A C G
Rea i n en adena de la polimerasa 4 P2R
#$ %$
8/12/2019 03 Fundamentos de Pcr
37/99
H S ntesis por DNA polimerasa
H - A T G C A T G C A T G C * *
A C G T- T A C G T
Rea i n en adena de la polimerasa 4 P2R
#$ %$
8/12/2019 03 Fundamentos de Pcr
38/99
H S ntesis por DNA polimerasa
H - A T G C A T G C A T G C * *
A C G T- T A C G T A
Rea i n en adena de la polimerasa 4 P2R
#$ %$
8/12/2019 03 Fundamentos de Pcr
39/99
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/
Rea i n en adena de la polimerasa 4 P2R
8/12/2019 03 Fundamentos de Pcr
40/99
Primero& la fusin separa las dos cadenas de DNA a alta
temperatura (01223C)
Rea i n en adena de la polimerasa 4 P2R
8/12/2019 03 Fundamentos de Pcr
41/99
Primero& la fusin separa las dos cadenas de DNA a alta
temperatura (01223C)
Rea i n en adena de la polimerasa 4 P2R
8/12/2019 03 Fundamentos de Pcr
42/99
Primero& la fusin separa las dos cadenas de DNA a alta
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
Rea i n en adena de la polimerasa 4 P2R
8/12/2019 03 Fundamentos de Pcr
43/99
Para controlar el proceso& necesitamos controlar la temperatura
7i repetimos el ciclo& tendremos cuatro copias
Rea i n en adena de la polimerasa 4 P2R
-usin8#3C
9ibridi!acin#2:;2
8/12/2019 03 Fundamentos de Pcr
44/99
&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
8/12/2019 03 Fundamentos de Pcr
48/99
@c E%itar las secuencias repetidas
#?#?
%?%?
#?#?%?
%?
D mero de primer
PCR%?repetido
#$:NNNNNNNNNNNNN@A@A:%$#$:NNNNNNNNNNNNN@A@A:%$
#$:NNNNNNNN@A@A:%$ %$:A@A@NNNNNNN
8/12/2019 03 Fundamentos de Pcr
49/99
@c E%itar las secuencias repetidas
#?#?
%?%?
no ha5 e tensin
PCR#? repetido
#?:@A@ANNNNNNNNNNNNN:%?#?:@A@ANNNNNNNNNNNNN:%?
#?:@A@ANNNNNNNN%?:NNNNNNNNA@A@:#?
#?#?
%?%?
8/12/2019 03 Fundamentos de Pcr
50/99
@c E%itar las secuencias repetidas
#?
#?
%?
%?
Productos de PCR no deseados
PCR-ormacin de hor"uillas
#$:NNNNNNNNNNN CA@ C:%$#$:NNNNNNNNNNNNNNNNN:%$
#$:NNNNNNNNNNN CA %$:C @
#?
%?
8/12/2019 03 Fundamentos de Pcr
51/99
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
8/12/2019 03 Fundamentos de Pcr
52/99
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
8/12/2019 03 Fundamentos de Pcr
53/99
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
8/12/2019 03 Fundamentos de Pcr
54/99
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
8/12/2019 03 Fundamentos de Pcr
55/99
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
8/12/2019 03 Fundamentos de Pcr
56/99
)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
8/12/2019 03 Fundamentos de Pcr
57/99
$ested PCR H A %eces 2 ronda de PCR no da un producto Wnico
product a partir de un templado comple8o5apareciendo un ;chorreado #llisDr>?ary @> #llis
HOB DO!S P2R BOR? 0HOB DO!S P2R BOR? 0
8/12/2019 03 Fundamentos de Pcr
79/99
HOB P2R 2A ! A@OC$ :>HOB P2R 2A ! A@OC$ :>
8/12/2019 03 Fundamentos de Pcr
80/99
@ $@ $ 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*)
HOB P2R 2A ! A@OC$ :>HOB P2R 2A ! A@OC$ :>
8/12/2019 03 Fundamentos de Pcr
81/99
@@
$%e first des ription of P2R t%eory$%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$HOB P2R 2A ! A@OC$..
8/12/2019 03 Fundamentos de Pcr
82/99
$%e first P2R e1periment$%e first P2R e1periment Dr> ? ell ?leppe ? ell ?leppe
8/12/2019 03 Fundamentos de Pcr
83/99
@ $@
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
(> $%ey belie6ed t%at t%e goal of in 6itro DNA synt%esis %adbeen a %ie6ed
'> Hig% temperat#re4stable DNA polymerases /ere #nkno/n
3> DNA loning + /%i % %ad #st beg#n+ be ame t%e preferredmet%od for prod# ing large amo#nts of DNA
,> aybe + be a#se of t%e diffi #lty to synt%esize large amo#ntsof primers no one t%o#g%t t%at t%e spe ifi amplifi ation ofgenomi DNA /as feasible
8/12/2019 03 Fundamentos de Pcr
84/99
HOB P2R 2A ! A@OC$ :>HOB P2R 2A ! A@OC$ :>
8/12/2019 03 Fundamentos de Pcr
85/99
@@
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
8/12/2019 03 Fundamentos de Pcr
86/99
HSaiki R+ ?>M S %arf SM "aloona "M #llis ?> @M Horn E> $M !rli % H>
8/12/2019 03 Fundamentos de Pcr
87/99
A>M Arn%eim N>+!nzymati amplifi ation of beta4globin genomi se7#en es and restri tion site analysis for diagnosis of si kle
Science + (8-5 De ')+ '3) @M "aloona "> A> Spe ifi synt%esis of DNA in 6itro 6ia a polymerase 4 atalyzed %ain rea tion> Methods in Enzymology + (8-* +(55 33545)>
HOB P2R 2A ! A@OC$ :>HOB P2R 2A ! A@OC$ :>
http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/034http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/034http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/004http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/004http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/004http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/007http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/007http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/005http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/005http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/005http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/005http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/005http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/005http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/007http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/007http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/007http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/004http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/004http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/004http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/034http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/034http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/0348/12/2019 03 Fundamentos de Pcr
88/99
@
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> $M #llis ?> @M !rli % HA>Primer4dire ted enzymati amplifi ation of DNA /it% a
t%ermostable DNA polymerase > Science + (8-- Fan'8+ '38
http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/009http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/009http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/009http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/009http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/009http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/009http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/009http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/009http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/009http://sunsite.berkeley.edu/cgi-bin/ebind2html/pcr/0098/12/2019 03 Fundamentos de Pcr
89/99
HOB P2R 2A ! A@OC$ :>HOB P2R 2A ! A@OC$ :>
8/12/2019 03 Fundamentos de Pcr
90/99
@
$%e Nobel Prize in 2%emistry (883
9"or ontrib#tions to t%ede6lopment of met%ods /it%inDNA4based %emistry:;
9"or %is in6ention of polymerase%ain rea tion
8/12/2019 03 Fundamentos de Pcr
91/99
'' Almost overnight( #C) became a standard research Almost overnight( #C) became a standard research
techni ue and practical applications soon &ollo$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
8/12/2019 03 Fundamentos de Pcr
92/99
Dete ting pat%ogens #sing genome4spe ifi primer pairs
Appli ations of P2R Appli ations of P2R
8/12/2019 03 Fundamentos de Pcr
93/99
.dentifi ation of geneti m#tations
$%e P2R te %ni7#e of
single strand con&ormational polymorphism *SSC#+sin gle strand con&ormational polymorphism *SSC#+
is one of t%e most /idely #sed met%ods for dete ting single base
pair %anges in genomi DNA]
]
Non4denat#ring a rylamide gelNon4denat#ring a rylamide gel
Appli ations of P2R Appli ations of P2R
8/12/2019 03 Fundamentos de Pcr
94/99
Eenotyping #sing kno/n se7#en e tagged sites
8/12/2019 03 Fundamentos de Pcr
95/99
Lab Appli ations of P2R Lab Appli ations of P2R
8/12/2019 03 Fundamentos de Pcr
96/99
Dire t loning from genomi DNA or DNA
,n vitro m#tagenesis and engineering of DNAHEeneration of spe ifi se7#en es of loned do#ble4stranded DNA for #se as probes
HEeneration of probes spe ifi for #n loned genes bysele ti6e amplifi ation of parti #lar segments of DNA
HEeneration of libraries of DNA from small amo#nts ofmRNA
HEeneration of large amo#nts of DNA for se7#en ing
8/12/2019 03 Fundamentos de Pcr
97/99
8/12/2019 03 Fundamentos de Pcr
98/99
irtual PCR ResultsH irtual PCR searches
entire genome lookingfor potential primersites .ithin 2G5GGG
ases of one another:H If found5 it performs a
%irtual PCR reaction:
http://grup.cribi.unipd.it/cgi-bin/mateo/vpcr2.cgihttp://grup.cribi.unipd.it/cgi-bin/mateo/vpcr2.cgi8/12/2019 03 Fundamentos de Pcr
99/99
Applications of PCR
H )utation testing5 e.g. cystic fi rosis:H #iagnosis or screening of ac7uired diseases5
e.g. AI#':H >enetic profiling in forensic5 legal and io-
di%ersity applications:
H 'ite-directed mutagenesis of genes:H ^uantitation of mR$A in cells or tissues: