PCR Parte 2 Completa

8/15/2019 PCR Parte 2 Completa

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PCR – Reaccion en cadena depolimerasa

Parte 2: METODOS


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PROGRAMA

La tecnica PCR

Variantes

Algunas aplicaciones


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ue tener en cuenta antes de utli!ar PCR?

$e necesitan las tecnicas moleculares% Cual%

"scala & tiempo y espacio fsiologica,ecologica, e'oluti'a!

Ni'el de discriminacion re(uerido

)ateriales a la mano

*iempo y dinero

In+ormacion de secuencias

*rabao en especies relacionadas

Laboratorio & in+raestructura, e(uipamiento,recursos


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E"uipamiento # recursos


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$unciona solamente con%i&lo'os moleculares?

Soporte de especialidadSoporte técnico-logístico

Personal de apoyo


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Re(isando el proceso


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)umero de copias


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*isuali!ando los resultados


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El pro%lema+++

Como identifcamos y detectamosuna secuencia especifca en un

genoma%


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El pro%lema+++

R"C-RDAR D-$ C-$A$ I)P-R*AN*"$#

.ay )/C.A$ secuencias di+erentes en un genoma

en las cuales no tenemos interes "$P"CI0ICIDAD! La cantidad del DNA en el (ue estamos

interesados en una muestra es )/1 pe(uenaA)PLI0ICACI-N!

Como identifcamos y detectamosuna secuencia especifca en un

genoma%!


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Re(ision: El tamano de los 'enomas

Pino# 23 billion bp

)aiz# 456 billion bp

$oya# 757 billion bp

.umano# 859 billion bp

)osca casera# :66 millionbp

Arroz# 966 million bp

"5 coli# 952 million bp

.IV# :5; miles bp

http://www.cbs.dtu.dk/databases/DOGS/abbr_table.txt

http://commtechlab.msu.edu/sites/dlc-me/zoo/Pf07002.jpg


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ue tan 'rande es ,+- %illones?

"l genoma ms5

Identifcar una secuencia de 466bp en un genoma seria comoencontrar una seccion deaproximadamente :6 cms delargo?


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Cuantas moleculas necesitamos para poder (erlas?

*en un gel de agarosa se necesita alrededor de 76ng de DNA

Para un productode 466 bp, (ue pesa 226 g@mol5bp,se necesitan 76e: @ 466B226! 8568e79 moles

Numero de A'ogadros 256EeE8

$e necesitaria 753e76 copies?

"n otras palabras, para poder

'isualizar un gen, el DNA en unamuestra de 766 celulas deberia sermultiplicado 736 millones de 'eces?????


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El pro%lema+++

Como identifcamos y detectamos unasecuencia especifca en un genoma%

R"C-RDAR D-$ C-$A$ I)P-R*AN*"$#

.ay )/C.A$ secuencias di+erentes en un genomaen las cuales no tenemos interes

La cantidad del DNA en el (ue estamos

interesados en una muestra es )/1 pe(uena

PCR resuel'e los dos aspectos???

S P E C I

I C I DA D

A ! P "

I I CA C I

O #


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PCR: los primeros ciclos


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*ariantes de PCR

)ested PCR


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R*PCR


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Análisis en tiempo real (real-time PCR)

Curvas de amplificación


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Al'unos usos de la PCR

Deteccion de DNA enmedicina +orense

Identifcacion de plantastransgenicas

Deteccion de in+eccion 'iral

Clonacion

Deteccion de DNA antiguo


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.sos de PCR: D)A anti'uo

http://www.$aseb.or%/opar/bloodsuppl&/pcr.ht'l

Arc/aeolo'ists /a(e /appil# sei!ed on PCR

and are appl#in' it in an ama!in' (ariet# of

0a#s+ 1t is /elpin' for e3ample to launc/ a

ne0 c/apter in t/e colorful and

contro(ersial stor# of t/e 24445#ear5old

Dead Sea Scrolls 0/ic/ are 0ritten on

parc/ment made out of s6ins from 'oats

and 'a!elles+ Researc/ers are anal#!in' t/e

parc/ment fra'ments to tr# to identif#

indi(idual animals t/e# came from+ T/e/ope is t/at t/e 'enetic information 0ill

'uide t/em in piecin' to'et/er t/e 74444

particles of scrolls t/at remain+


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.sos de PCR: Deteccion de enfermedades


PCR can also %e more accurate t/an standard

tests+ 1t is ma6in' a difference for e3ample in a

painful serious and often stu%%orn misfortune

of c/ild/ood t/e middle ear infection 6no0n as

otitis media+ T/e tec/ni"ue /as detected%acterial D)A in c/ildren8s middle ear fluid

si'nalin' an acti(e infection e(en 0/en culture

met/ods failed to detect it+ 9#me disease t/e

painful oint inflammation caused %# %acteria

transmitted t/rou'/ tic6 %ites is usuall#dia'nosed on t/e %asis of s#mptom patterns+ ;ut

PCR can !ero in on t/e disease or'anism8s D)A

contained in oint fluid permittin' speed#

treatment t/at can pre(ent serious

complications+


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.sos de PCR: Especies en peli'ro


Researc/ers /a(e used t/e tec/ni"ue to aid in

reducin' ille'al trade in endan'ered species and

products made from t/em+ ;ecause PCR is arelati(el# lo05cost and porta%le tec/nolo'# and

li6el# to %ecome more so it is adapta%le for field

studies of all 6inds in t/e de(elopin' countries+

1t is also a tool for monitorin' t/e release of

'eneticall# en'ineered or'anisms into t/een(ironment+


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.sos de PCR: D)A en ciencias forenses


T/e tec/ni"ue8s unparallelled a%ilit# to identif#

and cop# t/e tiniest amounts of e(en old and

dama'ed D)A /as pro(ed e3ceptionall#(alua%le in t/e la0 especiall# t/e criminal la0+

PCR is an indispensa%le adunct to forensic

D)A t#pin'5commonl# called D)A

fin'erprintin'+


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.sos de PCR: Pro%ando inocencia


D)A t#pin' is onl# one of man# pieces of

e(idence t/at can lead to a con(iction %ut it /as

pro(ed in(alua%le in demonstratin' innocence+

Do!ens of suc/ cases /a(e in(ol(ed people 0/o

/a(e spent #ears in ail for crimes t/e# did notcommit+ One e3ample is


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T/e met/od is especiall# useful for searc/in' out

disease or'anisms t/at are difficult or impossi%le

to culture suc/ as man# 6inds of %acteria fun'i

and (iruses %ecause it can 'enerate anal#!a%le

"uantities of t/e or'anism8s 'enetic material foridentification+ 1t can for e3ample detect t/e

A1DS (irus sooner durin' t/e first fe0 0ee6s

after infection t/an t/e standard E91SA test+

PCR loo6s directl# for t/e (irus8s uni"ue D)A

instead of t/e met/od emplo#ed %# t/e standardtest 0/ic/ loo6s for indirect e(idence t/at t/e

(irus is present %# searc/in' for anti%odies t/e

%od# /as made a'ainst it+


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.sos de PCR: D)A anti'uo


Arc/aeolo'ists are findin' t/at PCR can

illuminate /uman cultural practices as 0ell

as /uman %iolo'#+ Anal#!in' pi'ments

from -4445#ear5old roc6 paintin's in Te3as

t/e# found one of t/e components to %e

D)A pro%a%l# from %ison+ T/e animals

did not li(e near t/e Pecos Ri(er at t/at

time so t/e paleo5artists must /a(e 'one to

some effort to o%tain suc/ an unusual

in'redient for t/eir paint+ Ta6in' so muc/trou%le su''ests t/at t/e paintin's 0ere not

simpl# decorations %ut /ad reli'ious or

ma'ical si'nificance+


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PCR can e(en dia'nose t/e diseases of t/e past+$ormer (ice president and presidential

candidate >u%ert >+ >ump/re# under0ent tests

for %ladder cancer in 7=@+ Alt/ou'/ t/e tests

0ere ne'ati(e /e died of t/e disease in 7=@+ 1n

7==- researc/ers compared a 7=@ tissue samplefrom /is cancer5ridden %ladder 0it/ /is 7=@

urine sample+ Bit/ t/e /elp of PCR

amplification of t/e small amount of D)A in t/e

2@5#ear5old urine t/e# found identical

mutations in t/e p, 'ene 0ell56no0n forsuppressin' tumors in %ot/ samples+

>ump/re#8s e3amination in 7=@ ma# /a(e

re(ealed t/e cancerous 'ro0t/ if t/e tec/ni"ues

of molecular %iolo'# 0ere as 0ell understood

t/en as t/e# /a(e %ecome t/e researc/ers said+


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Aplicaciones

"emplo de un caso decriminalFstica resueltoutilizando electro+oresis engel 'ertical de acrilamida5

$i se obser'an los patronesde bandas podrGcomprobarse como losperfles obtenidos en lasmanc


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.tilidad de PCR

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PCR Parte 2 Completa