metode de ggstudiu a genelor
TRANSCRIPT
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Metode deanaliză a genelor
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Expresia genelor
ADN
ARNm
Proteină
Caracter 5’-ATTGCAAGATTACCATGT-’ Catena codogenă !netranscrisă"
’-TAACGTTCTAATGGTACA-5’ Catena anticodogenă !transcrisă"
Transcripţie
5’-A##GCAAGA##ACCA#G#-’ ARNm
Translaţie
$e% & Ala & Arg & $e% & Pro & C's polipeptid
Maturizare
Caracter (enotipic normal
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Expresia genelor
ADNm%t
ARNmm%t
Proteinăan
Caracter an
5’-ATTACAAGATTACCATGT-’ m%ta)ie G*+A’-TAATGTTCTAATGGTACA-5’
Transcripţie
5’-A##ACAAGA##ACCA#G#-’ ARNmm%t
Translaţie
$e% & T,r & Arg & $e% & Pro & C's polipeptidan
Caracter (enotipic anormal
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Analiza genelor
ec.en)iere
PCR
o%t,ern-/lot
0i/ridare in situ
ADN ProteineARN
Nort,ern-/lot
RT&PCR
0i/ridare in situ
1estern-/lot
ec.en)iere
Analiza (%nc)iei
Analiza,istoc,imică
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2/)inerea 3R & (ragmentelor de restric)ie prinac)i%nea speci(ică a ER !enzimelor de restric)ie"
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RFLPs – polimorfismul FR la două persoane X şi Z
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M
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4
6 7/
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: 7/
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9 7/
5 7/
:;5 7/
:;5 7/
6 7/
9 7/
5 7/
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Etapele te,nicii PCR
• Denat%rarea ADN la <9oC
A= Pregătirea amestec%l%i c% toate componentele reactant
• Răcirea p>nă la temperat%ra de renat%rare a primerilor • Elongarea catenelor ADN la ?:oC
@= Repetarea a%tomată a proced%rilor de denat%rare-renat%rare-elongare de 6-5 ori
C= Analiza prod%ilor PCR - electro(oreza
- colorarea - interpretarea rez%ltatelor
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Extragerea ADN Pregătireacomponentelor pentr%
PCR
Ampli(icarea
ADN-)intă
Electro(oreza
prod%ilor PCR
Colorarea i
.iz%alizarea prod%ilorPCR
Bnterpretarea
rez%ltatelor
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Identificarea inserţiilor / deleţiilor
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Identificarea mutaţiilor punctiforme
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Identificarea agenţilor infecţioşi
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Stabilirea paternităţii filiaţiei!
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Optimizarea condiţiilor de desfăşurare a PCR
"ependenţa cantităţii de produse amplificate de concentraţia #"$ genomic utili%ată &n P'R(S)a utili%at #"$ &n cantitate de* + – ,-. g- 0 – ,-0. g- 1 – ,-+ g- 2 – ,-,0 g
'oncentraţiile critice ale substanţelor pentru in3ibarea acti4ităţii 5a6)polimera%ei
Substanţa 'oncentraţia critică
SDS >0.005% (m/v)
Fenol >0.2% (v/v)
Etanol >1% (v/v)
Isopropanol >1% (v/v)
CH3COOa >5 m!
aCl >25 m!
ED"# >0.5 m!
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Optimizarea condiţiilor dedesfăşurare a PCR
'aracteristica primerilor utili%aţi &n P'R
7ena Sec4enţa Lungimea- ba%e 8 79' 5m- o'
#': 5$C"&&#&#CC#C"CCC#"CC"""C"3$ 2' 5' 5
5$&#"&"&&CC#"C#C#""C&"C#&#"3$ 25 ' 5
#5+ R 5$C"&&C"CC""&C"&&"&"&&3$ 20 *5 5
5$&&&C##&C&"#"#""""C"&&"&3$ 23 ' 55
$;S 5+##&&C#&&#&#C#&"&&#"&+ 22 55 5,
5+CCC#&"C##"CCC"""&&"&C"C#3+ 2' 5' 5
"ependenţa cantităţii produselor P'R de temperatura de renaturare a primerilor(<n condiţiile P'R s)a utili%at* + – to' optimală ).o'= 0 – to' optimală= 1 – to' optimală 9.o'(
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Optimizarea condiţiilor de desfăşurare a PCR
"ependenţa cantităţii produselor P'R de concentraţia ionilor >g09(
'oncentraţia utili%ată de >g'l0* + – 0-.m>= 0 – .m>(
"ependenţa cantităţii de #"$ amplificat de numărul de cicluri P'R(+ – 1, cicluri= 0 – 1. cicluri= 3 – 40 cicluri.
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Analiza polimorfismului I/D pentru ena AC!
#nali%a electroforetică &n gel de agaro%ă de 08 a produselor P'R re%ultatedin amplificarea #"$ uman cu primeri corespun%ători genei #':(
> – marc3er al lungimii= + – genotip I/"= 0 – genotip "/"= 1 – genotip I/I(
I
D
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Analiza polimorfismului "/T pentru ena #O$
! 1 2 3
Re%ultatele amplificării #"$ uman cu primeri corespun%ători genei $;S(> – marc3er al lungimii= + – genotip 7/7= 0 – genotip 5/5= 1 – genotip 7/5(
G
T
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Analiza polimorfismului A/C pentru ena AT% R
Re%ultatele amplificării #"$ uman cu primeri corespun%ătorigenei #5+ R digerate cu a?utorul en%imei "de I(
> – marc3er al lungimii= + – genotip #/#= 0 – genotip #/'
A
C
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Te&nica Real'time'PCR
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ARM$'PCR
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5e3nica Sout3ern)blot
:@tragerea #"$ Restriţia #"$ :lectrofore%a #"$
"enaturarea şitransferul #"$pe membrane
#utoradiografiaAibridare cu sonda
Bi%uali%area şiinterpretarea
re%ultatelor
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R()Ps şi analiza enotipului
!lectroforeza (R a * persoane A+, şi C
Alela #ormală cu * $R Alela mutantă cu - $R+ mutaţie .n $R-
@ !Nm"
C !mm"
A !NN"
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5e3nica $ort3ern)blot
:@tragerea #R$ :lectrofore%a #R$
5ransferul #R$pe membrane
#utoradiografiaAibridare cu sonda
Bi%uali%area şiinterpretarea
re%ultatelor
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Bdenti(icarea ni.el%l%ide expresie i a .arianteide splicing
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5’-ATTACAAGATTACCATGT-’ catena codogenă’-TAATGTTCTAATGGTACA-5’ catena anticodogenă matriţă0
’-TAATGTTCTAATGGTACA-5’5’-ATTAC-’
’-TAATGTTCTAATGGTACA-5’5’-ATTACAA-’
’-TAATGTTCTAATGGTACA-5’5’-ATTACAAG-’
’-TAATGTTCTAATGGTACA-5’5’-ATTACAAGA-’
’-TAATGTTCTAATGGTACA-5’5’-ATTACAAGATT-’
’-TAATGTTCTAATGGTACA-5’5’-ATTACA-’
’-TAATGTTCTAATGGTACA-5’5’-ATTACAAGAT-’
A; T; C; T & ddNTP !:’;’-ddNTP"
pentru stoparea sintezei
5’-ATTA -- primer marcat P*-
pentru AD#'polimerază
A; T; C; T & dNTP !:’-dNTP"pentru sinteza noilor catene
5’-ATTACA-’
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5’-ATTACAA-’
5’-ATTACAAG-’
5’-ATTACAAGA-’
5’-ATTACAAGATT-’
5 -ATTACA-
5’-ATTACAAGAT-’
5’-ATTACAAGATTA-’
5’-ATTACAAGATTACCA-’
5’-ATTACAAGATTACCAT-’
5’-ATTACAAGATTACCATG-’
5’-ATTACAAGATTACCATGT-’
1
'A T C G
5’-ATTAC-’
5’-ATTACAAGATTAC-’
5’-ATTACAAGATTACC-’ 5’-ATTACAAGATTACCATGT-’
#
5
7
'
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'
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1
10 23 TAAATGGCTA4444444*3'0
Metoda a%tomată
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Metoda a%tomată
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0i/ridarea in situ c%%tilizarea preparatelor
de cromozomi meta(azici
0i/ridarea in situ c%%tilizarea preparatelor
c% n%clei inter(azici
0i/ridarea in situ pentr%depistarea sec.en)elor ADN
Aib id i it
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Aibridareain situ
cuutili%area probei sens stCnga!şi antisens dreapta!
Aibridarea in situ pentru depistareasec4enţelor #R$
#nali%a3istoc3imică –identificarea
proteinelor &n ţesut
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