Amplification of terminal ends of Newcastle disease virus genome by rapid amplification of complementary deoxyribonucleic acid ends.
4*INIIRNAIIONAI,
O()NIERINO]
ON
BIOIECHNOTO
BIOSCIINCIS
AND
PROGRAITI
ANDABSTRACT
BOOK
ICBB-Bqli
Rofl,leptcmbcr 20 - ?,1,201|2
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CIGAFETESMOKE
EXPOSURE
DECSEASE
SPCRM
QUALIIYOFMALEMICE
lMusMUscuLUsL,l
NIMADERAISUARNI,
A.A.5,A.5UXMANING5IH,
IRIANISETYAWATI
BiolosyDepertement,
Facultyof MathemaucandMtural Sciences,
Ud.yan.University,Bali
Abstract
Th€ eff€ctsot cisarettesmokeon severalaspectsspermqualityof male
rr'i.e(Musnusculusl.l h.d beenobserued.
Ten male mice,10'11 weeks
old exposureby ten cigar€ttermoke, labeledCountryeveryday for 36
d a y sa n dt e n m a l em i c ef o r c o n v o lS. p e r mws e r e o b t a i . efdr o mt h ec a u d a l
part of epldidymis.
Theresultsrhowedthat ciSaretteexposur€decre.seor
incfeaseof the
sp€rmqu.lity, i.e. decreat€th€ amolJnlof spermatozoa,
imnotile deadandab.ormalrDermar02
oa
Keywotd : cigotette tftoke. cauclolepididymyt,tpem quolity
P-3-t6
adplitietions ot T.rminalEndsof N.wc.sile OLea3eViru! Genomcby
8.pid adplific.tion of Complementa.yD@ry.ibon!.lei. Acid E.dt
ANAI(AGUNGA''UMIMII AOI, NYOMANMANTI(AsTAWA"
YAsUNOAU
MATSUMOTO!
!PatholoByLaboratory':virolosyLaboratory,
Fac!ltyofveterinary
Medicine,
u d a y a nuan i v e r s l i y ' l l nP.I S u d i r m aOne n p a s B
a ra l i .
3
Laboratoryof GlobalAhimalRerource
Sciefce,Depanm€ntof 6loba
Graduate
Schoolof Agric!lturaland Lfe 5ciences,
the
furic!ltora sciences,
Unlve15ltY
otTokyo,
TokVo,
Japan.
l-1-1 Yayoi,Bunkyo.ku,
Co(esponding
a!ihor:mirah638@yahoo.co.id
Abrkact
Leaderand trailer (termlnalends)of nbonucleicacid (RNA)seqleicesis
importantin characteriu
ing nov€lParamyxovirus
astheycontainlmportant
sigoalsfor repllcationand transcnptbntheir Senomes and therefore
importantin understanding
the processof viral evolutron.Conventional
polymerasechain r€action{PCR)ls normaly !iable to amplit these
udayin: U^ee6ity,Bai, ?u2 s.pr.mb!i 20rI
4rh hrsn.rionll Conrd.ft. o. 3ioni.n.6 rnd Siotednololy
^d€o.in3r'r.*i.nc.!tdh..lh
(anr 4iryd.v.roph!nrin pai .lrrd rnd food:.(uriry
regions.Recently,
rapldlmplificationof CDNAEndsIRACE)
is a pcR-based
iechniquewhich hasb€endevelopedto amplifytheseregioB in orderto
determineRNA terminal sequences,
Most RACEm€thodr reportedin
Iterature, use differentreagentfor 5lRACEand 3 -RACE,
are thereforc
costly.In this study,the leader(sL CDNAendt andtrailer(3! CDNAeids)
of the vira RNAgenomeof NewcatledGeaseVlru3(NDv)/Bali./07
were
ahplified.The 5' GDNAendsof the varalRNAwe.€ litated with a 3 end
cordecypin-blocked
adaptor DT88 L[ing f4 RNA ligase.The resutting
ad:pto.lltated cDNAswas then amplifiedustnethe primer DT89and
primerspeciiicfor Nucleoprotein
gen€of NOV/8at-1l07asa forwardand
revers€primersrespectivelv.
polydATP-tailed
Th€3'- CDNAendswere
and
polydcTP-tailed
using14 terminaldeory.ucleotidyltransferase.
The PCR
reactionw.s performedusingthe templatesof both the polvdATP-tailed
and polydcTP-tailed
CDNAand wee performedIndependently
Lrsing
the
samefoMard prlmerspecificLargepolyrneras€
proteingeneand reveEe
pramer olitoldT) and oligo-(dc) primer respedtue;y.The obtained
amplicons
werethenclonedand positiveclonesweresequenced,
Withthis
methodbothleaderandtraier of NDV/Bali1/07canbe amplified,without
l s i n ga h t h l y c o s tR A CkEt .
Keyword:genome,leader,trailer,RACE
r.rdayaf
. univ.6iy,saI, r1 r2 5.pr.mber?012
.i.n..r rid 8iol..i.o ocv
toods..unrv
Advri.in! ir...'en..slorh€alth {.ntiaSid3d.vdoPn.dt n pa{ cuLa4and
Faclltyof Medicine,Sebelaj
P-3-8
DePannentof Mi.robiolo8y, r!c33p@.!!-!a!.!s
Facultyof Medicine,Sebelas
Maret univesity, ll. lr.
364, Sumkana
slr.ni
o.pa.tmenrot Microblolo8y, !!3!3!.@tdpqrp
F*uhy ol Medicine,5€b.1.5 a
M..er u.ivertiy, n- lr.
36A, 5!6lana
Slt.oi
P.3.10
P.3.11
oeradnenrof Mic.oblolo8v,anea.o@vahoo,co
Fe.uliyot M€dicine,Sebebs !!1
Maret Unive6itt, ll. lr.
Sutami 36A, surakafta
57126,Indonesia
Depanhent oi analyst of Aryadnvani35@q
He.lth, stikes wna M€dika 431!!E!
P-3.12
!!-l!!!idt!!!@I.
!g!.r!.lc
P.!-13
Medi.in€,0enpa$r,3ari,lndo
!!r44.a$@!!
s.iences UdavanaUnive6ityP.3-15
P.1.16
BloloSyDepenenent, faculty
ot MarhenaticandN!tural
'vl.olo8vLaboatory,tacuhy
ICEEE|ifOT_
Udry...|Jni4dtv,3a|.'2122s.plmb{2012
3438!Mt4!rk
9r!
O()NIERINO]
ON
BIOIECHNOTO
BIOSCIINCIS
AND
PROGRAITI
ANDABSTRACT
BOOK
ICBB-Bqli
Rofl,leptcmbcr 20 - ?,1,201|2
ITir'liilas
maal@
d(bodaasff
[l
Ad@n.isrire$i!nar rorh.ahh(rntl,siDsd.vdopm.niin prtkulaa aid roodre.rry
CIGAFETESMOKE
EXPOSURE
DECSEASE
SPCRM
QUALIIYOFMALEMICE
lMusMUscuLUsL,l
NIMADERAISUARNI,
A.A.5,A.5UXMANING5IH,
IRIANISETYAWATI
BiolosyDepertement,
Facultyof MathemaucandMtural Sciences,
Ud.yan.University,Bali
Abstract
Th€ eff€ctsot cisarettesmokeon severalaspectsspermqualityof male
rr'i.e(Musnusculusl.l h.d beenobserued.
Ten male mice,10'11 weeks
old exposureby ten cigar€ttermoke, labeledCountryeveryday for 36
d a y sa n dt e n m a l em i c ef o r c o n v o lS. p e r mws e r e o b t a i . efdr o mt h ec a u d a l
part of epldidymis.
Theresultsrhowedthat ciSaretteexposur€decre.seor
incfeaseof the
sp€rmqu.lity, i.e. decreat€th€ amolJnlof spermatozoa,
imnotile deadandab.ormalrDermar02
oa
Keywotd : cigotette tftoke. cauclolepididymyt,tpem quolity
P-3-t6
adplitietions ot T.rminalEndsof N.wc.sile OLea3eViru! Genomcby
8.pid adplific.tion of Complementa.yD@ry.ibon!.lei. Acid E.dt
ANAI(AGUNGA''UMIMII AOI, NYOMANMANTI(AsTAWA"
YAsUNOAU
MATSUMOTO!
!PatholoByLaboratory':virolosyLaboratory,
Fac!ltyofveterinary
Medicine,
u d a y a nuan i v e r s l i y ' l l nP.I S u d i r m aOne n p a s B
a ra l i .
3
Laboratoryof GlobalAhimalRerource
Sciefce,Depanm€ntof 6loba
Graduate
Schoolof Agric!lturaland Lfe 5ciences,
the
furic!ltora sciences,
Unlve15ltY
otTokyo,
TokVo,
Japan.
l-1-1 Yayoi,Bunkyo.ku,
Co(esponding
a!ihor:mirah638@yahoo.co.id
Abrkact
Leaderand trailer (termlnalends)of nbonucleicacid (RNA)seqleicesis
importantin characteriu
ing nov€lParamyxovirus
astheycontainlmportant
sigoalsfor repllcationand transcnptbntheir Senomes and therefore
importantin understanding
the processof viral evolutron.Conventional
polymerasechain r€action{PCR)ls normaly !iable to amplit these
udayin: U^ee6ity,Bai, ?u2 s.pr.mb!i 20rI
4rh hrsn.rionll Conrd.ft. o. 3ioni.n.6 rnd Siotednololy
^d€o.in3r'r.*i.nc.!tdh..lh
(anr 4iryd.v.roph!nrin pai .lrrd rnd food:.(uriry
regions.Recently,
rapldlmplificationof CDNAEndsIRACE)
is a pcR-based
iechniquewhich hasb€endevelopedto amplifytheseregioB in orderto
determineRNA terminal sequences,
Most RACEm€thodr reportedin
Iterature, use differentreagentfor 5lRACEand 3 -RACE,
are thereforc
costly.In this study,the leader(sL CDNAendt andtrailer(3! CDNAeids)
of the vira RNAgenomeof NewcatledGeaseVlru3(NDv)/Bali./07
were
ahplified.The 5' GDNAendsof the varalRNAwe.€ litated with a 3 end
cordecypin-blocked
adaptor DT88 L[ing f4 RNA ligase.The resutting
ad:pto.lltated cDNAswas then amplifiedustnethe primer DT89and
primerspeciiicfor Nucleoprotein
gen€of NOV/8at-1l07asa forwardand
revers€primersrespectivelv.
polydATP-tailed
Th€3'- CDNAendswere
and
polydcTP-tailed
using14 terminaldeory.ucleotidyltransferase.
The PCR
reactionw.s performedusingthe templatesof both the polvdATP-tailed
and polydcTP-tailed
CDNAand wee performedIndependently
Lrsing
the
samefoMard prlmerspecificLargepolyrneras€
proteingeneand reveEe
pramer olitoldT) and oligo-(dc) primer respedtue;y.The obtained
amplicons
werethenclonedand positiveclonesweresequenced,
Withthis
methodbothleaderandtraier of NDV/Bali1/07canbe amplified,without
l s i n ga h t h l y c o s tR A CkEt .
Keyword:genome,leader,trailer,RACE
r.rdayaf
. univ.6iy,saI, r1 r2 5.pr.mber?012
.i.n..r rid 8iol..i.o ocv
toods..unrv
Advri.in! ir...'en..slorh€alth {.ntiaSid3d.vdoPn.dt n pa{ cuLa4and
Faclltyof Medicine,Sebelaj
P-3-8
DePannentof Mi.robiolo8y, r!c33p@.!!-!a!.!s
Facultyof Medicine,Sebelas
Maret univesity, ll. lr.
364, Sumkana
slr.ni
o.pa.tmenrot Microblolo8y, !!3!3!.@tdpqrp
F*uhy ol Medicine,5€b.1.5 a
M..er u.ivertiy, n- lr.
36A, 5!6lana
Slt.oi
P.3.10
P.3.11
oeradnenrof Mic.oblolo8v,anea.o@vahoo,co
Fe.uliyot M€dicine,Sebebs !!1
Maret Unive6itt, ll. lr.
Sutami 36A, surakafta
57126,Indonesia
Depanhent oi analyst of Aryadnvani35@q
He.lth, stikes wna M€dika 431!!E!
P-3.12
!!-l!!!idt!!!@I.
!g!.r!.lc
P.!-13
Medi.in€,0enpa$r,3ari,lndo
!!r44.a$@!!
s.iences UdavanaUnive6ityP.3-15
P.1.16
BloloSyDepenenent, faculty
ot MarhenaticandN!tural
'vl.olo8vLaboatory,tacuhy
ICEEE|ifOT_
Udry...|Jni4dtv,3a|.'2122s.plmb{2012
3438!Mt4!rk
9r!