lezione snupe 1
TRANSCRIPT
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Università degli Studi di Firenze Facoltà di Scienze Matematiche Fisiche e Naturali
Corso di Laurea in Biotecnologie
Dott. Ferri Lorenzo Dott. Sparvoli Valentina
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Single Nucleotide Primer Extension
Permette di rilevare polimorfismi puntiformi (SNPs) in qualsiasi punto di una regione del
DNA amplificabile via PCR.
Attraverso l’estensione di uno o più primerdi un solo nucleotide complementare alla base polimorfica che si vuole studiare.
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T5’ 3’
A 5’3’T
Single Nucleotide Primer Extension
O BASE
H
H
H
H HH
CH2
O-PO
OPO
OO-
PO
OO-
O-
I
lunghezza
ELETTROFORESI
CAPILLARE
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ANALISI SNuPE
DETERMINAZIONE GENOTIPI
Single Nucleotide Primer Extension
INDIVIDUAZIONE MUTAZIONI
IDENTIFICAZIONE di SPECIE
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PROTOCOLLO SPERIMENTALE
1. ANALISI BIONFORMATICARicerca e recupero, per ogni specie, delle sequenze nucleotidiche del MARCATORE MOLECOLARE (es.
un gene) scelto come bersaglio dell’analisi, disponibili in banca dati
SPECIE ASPECIE B
SPECIE CAF108056 AF108057 AF269041 AY074260 AY460027 ........
AY473021 AF057108 AY604126 AY067403 AF269017 ........
AF247043 AF604821 AY600626 AY744103 AY690319 ........
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PROTOCOLLO SPERIMENTALE
1. ANALISI BIONFORMATICAAllineamento delle sequenze recuperate per ogni specie
AF108056 Specie A AGTCAATCGACCGTTCGGACCGGTGACTATACGACG
AF108057 Specie A AGTCAACCGACCGTTCCGACCGGTGACTATTCGACG
AF269041 Specie A AGTCAATCGACCGTTCGGACCGGTGACTATACGACG
AY074260 Specie A AGTCAATCGGCCGTTCGGACCGCTGACTATACGACG
AY460027 Specie A AGTCAATCGGCCGTTCGGCCCGCTGACTATACGACG
Creazione di SEQUENZE CONSENSO
Consensus sequence AGTCAAYCGRCCGTTCGGCCCGSTGACTATWCGACG
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PROTOCOLLO SPERIMENTALE
1. ANALISI BIONFORMATICAAllineamento delle sequenze
consenso delle specie considerate
SpecieA 95 AGTCAAYCGRCCGTTCGCCCGCTGACTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieB 95 AGTCAAYCGACCGTTCCACCGCTGAGTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieC 95 AGTCAAYCGGCCGTTCGACCGTTGAGTATACGGCGTCAGTAGGCACTYAYCCGACTRAAT
SpecieA 155 ACTTYCGGGCAATCTACGRYYTCAAYCGRCAGTTCGGRTCACGACGACGTCYTRCAGTTC
SpecieB 155 ACYTYCGRGCAATCAACGRYCTCAAYCGRCAGTYCGGRTCACGACGACGTCYTRCAGTTC
SpecieC 155 ACTTYCGGGCAATCGACGRYYTCAATCGRCCGTTCGGATCACGACTACGTCYTRCAGTTC
SpecieA 215 TCTACGTRCGGTCACGACGTCGCTGAGTATTCGTCYTRCAGMCCGTTCCACCGCACGACG
SpecieB 215 TCTACGTRCAGTGACGACGTCGCTGAGTATGCGTCYTRCAGACCGGTCCACCGCRCGACG
SpecieC 215 TCTACGTRCAGTCACGACGTCGCTGAGTATACGTCYTRCAGACCGGTCCACCGCACGACG
Individuazione SNPs SPECIE SPECIFICI
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PROTOCOLLO SPERIMENTALE
2. DISEGNO PRIMER
CRITERI
Devono ricadere all’interno di una regione amplificabile via PCR
Scelta SNPs utili per l’analisi SNuPE
Devono presentare regioni conservate a monte o a valle
Singolarmente o combinati insieme devono consentire di discriminare le specie
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PROTOCOLLO SPERIMENTALE
2. DISEGNO PRIMER
SpecieA 95 AGTCAAYCGRCCGTTCGCCCGCTGACTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieB 95 AGTCAAYCGACCGTTCCACCGCTGAGTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieC 95 AGTCAAYCGGCCGTTCGACCGTTGAGTATACGGCGTCAGTAGGCACTYAYCCGACTRAAT
SpecieA 155 ACTTYCGGGCAATCTACGRYYTCAAYCGRCAGTTCGGRTCACGACGACGTCYTRCAGTTC
SpecieB 155 ACYTYCGRGCAATCAACGRYCTCAAYCGRCAGTYCGGRTCACGACGACGTCYTRCAGTTC
SpecieC 155 ACTTYCGGGCAATCGACGRYYTCAATCGRCCGTTCGGATCACGACTACGTCYTRCAGTTC
SpecieA 215 TCTACGTRCGGTCACGACGTCGCTGAGTATTCGTCYTRCAGMCCGTTCCACCGCACGACG
SpecieB 215 TCTACGTRCAGTGACGACGTCGCTGAGTATGCGTCYTRCAGACCGGTCCACCGCRCGACG
SpecieC 215 TCTACGTRCAGTCACGACGTCGCTGAGTATACGTCYTRCAGACCGGTCCACCGCACGACG
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PROTOCOLLO SPERIMENTALE
2. DISEGNO PRIMERCARATTERISTICHE
Annealing su tutte le specieDisegno primer per
l’analisi SNuPE Coda di poli T all’estremità 5’
SpecieA 215 TCTACGTRCGGTCACGACGTCGCTGAGTATTCGTCYTRCAGMCCGTTCCACCGCACGACG
SpecieB 215 TCTACGTRCAGTGACGACGTCGCTGAGTATGCGTCYTRCAGACCGGTCCACCGCRCGACG
SpecieC 215 TCTACGTRCAGTCACGACGTCGCTGAGTATACGTCYTRCAGACCGGTCCACCGCACGACG
5’-(T)5AGTCACGACGTCGCTGAGTAT-3’
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PROTOCOLLO SPERIMENTALE
3. PCRFw Rev
245
PURIFICAZIONE PCR
1. Marker; 2. campione; 3. controllo negativo
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PROTOCOLLO SPERIMENTALE
4. REAZIONE SNuPE
PRODOTTO di PCR PURIFICATO
PRIMER SNuPEREADY REACTION MIX
AmpliTaq®DNA Polymerase;FS Reaction Buffer;
ddNTPs (ddGTP,ddCTP,ddTTP,ddATP)
O BASE
H
H
H
H HH
CH2
O-PO
OPO
OO-
PO
OO-
O-
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PROTOCOLLO SPERIMENTALE
4. REAZIONE SNuPE (SIMPLEX)
DenaturazioneDenaturazione..T5’ 3’Specie ASpecie AA 5’3’
AnnealingAnnealingdel primer.del primer. T5’ 3’
A 5’3’Specie ASpecie A
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STRATEGIA SPERIMENTALE
4. REAZIONE SNuPE (SIMPLEX)
Estensione del Estensione del primer.primer.
T5’ 3’
A 5’3’T
O BASE
H
H
H
H HH
CH2
O-PO
OPO
OO-
PO
OO-
O-
Specie ASpecie A
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PROTOCOLLO SPERIMENTALE
5. ELETTROFORESI CAPILLARE (SIMPLEX)
Elettroforesi capillare.Elettroforesi capillare.
I
lunghezza
I
lunghezza
I
lunghezza
3’ 5’TSpecie CSpecie C
Specie BSpecie B 3’ C 5’
Specie ASpecie A 3’ A 5’
G
A
T
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PROTOCOLLO SPERIMENTALE
5. ELETTROFORESI CAPILLARE (SIMPLEX)
Elettroforesi capillare Elettroforesi capillare
I
3’ 5’TA
Specie BSpecie B
Specie ASpecie A 3’ AT
5’
lunghezza
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PROTOCOLLO SPERIMENTALE
SpecieA 95 AGTCAAYCGRCCGTTCGCCCGCTGACTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieB 95 AGTCAAYCGACCGTTCCACCGCTGAGTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieC 95 AGTCAAYCGGCCGTTCGACCGTTGAGTATACGGCGTCAGTAGGCACTYAYCCGACTRAAT
SpecieA 155 ACTTYCGGGCAATCTACGRYYTCAAYCGRCAGTTCGGRTCACGACGACGTCYTRCAGTTC
SpecieB 155 ACYTYCGRGCAATCAACGRYCTCAAYCGRCAGTYCGGRTCACGACGACGTCYTRCAGTTC
SpecieC 155 ACTTYCGGGCAATCGACGRYYTCAATCGRCCGTTCGGATCACGACTACGTCYTRCAGTTC
SpecieA 215 TCTACGTACAGTCACGACCTCGCTGAGTATTCGTCYTRCAGMCCGTTCCACCGCACGACG
SpecieB 215 TCTACGTACAGTGACGACTTCGCTGAGTATGCGTCYTRCAGACCGGTCCACCGCRCGACG
SpecieC 215 TCTACGTACAGTCACGACTTCGCTGAGTATACGTCYTRCAGACCGGTCCACCGCACGACG
TGSpecie C
TASpecie B
CASpecie A
SNP 2SNP 1Specie
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PROTOCOLLO SPERIMENTALE
SpecieA 95 AGTCAAYCGRCCGTTCGCCCGCTGACTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieB 95 AGTCAAYCGACCGTTCCACCGCTGAGTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieC 95 AGTCAAYCGGCCGTTCGACCGTTGAGTATACGGCGTCAGTAGGCACTYAYCCGACTRAAT
SpecieA 155 ACTTYCGGGCAATCTACGRYYTCAAYCGRCAGTTCGGRTCACGACGACGTCYTRCAGTTC
SpecieB 155 ACYTYCGRGCAATCAACGRYCTCAAYCGRCAGTYCGGRTCACGACGACGTCYTRCAGTTC
SpecieC 155 ACTTYCGGGCAATCGACGRYYTCAATCGRCCGTTCGGATCACGACTACGTCYTRCAGTTC
SpecieA 215 TCTACGTACAGTCACGACCTCGCTGAGTATTCGTCYTRCAGMCCGTTCCACCGCACGACG
SpecieB 215 TCTACGTACAGTGACGACTTCGCTGAGTATGCGTCYTRCAGACCGGTCCACCGCRCGACG
SpecieC 215 TCTACGTACAGTCACGACTTCGCTGAGTATACGTCYTRCAGACCGGTCCACCGCACGACG
TGSpecie C
TASpecie B
CASpecie A
SNP 2SNP 1Specie
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PROTOCOLLO SPERIMENTALE
SpecieA 95 AGTCAAYCGRCCGTTCGCCCGCTGACTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieB 95 AGTCAAYCGACCGTTCCACCGCTGAGTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieC 95 AGTCAAYCGGCCGTTCGACCGTTGAGTATACGGCGTCAGTAGGCACTYAYCCGACTRAAT
SpecieA 155 ACTTYCGGGCAATCTACGRYYTCAAYCGRCAGTTCGGRTCACGACGACGTCYTRCAGTTC
SpecieB 155 ACYTYCGRGCAATCAACGRYCTCAAYCGRCAGTYCGGRTCACGACGACGTCYTRCAGTTC
SpecieC 155 ACTTYCGGGCAATCGACGRYYTCAATCGRCCGTTCGGATCACGACTACGTCYTRCAGTTC
SpecieA 215 TCTACGTACAGTCACGACCTCGCTGAGTATTCGTCYTRCAGMCCGTTCCACCGCACGACG
SpecieB 215 TCTACGTACAGTGACGACTTCGCTGAGTATGCGTCYTRCAGACCGGTCCACCGCRCGACG
SpecieC 215 TCTACGTACAGTCACGACTTCGCTGAGTATACGTCYTRCAGACCGGTCCACCGCACGACG
TGSpecie C
TASpecie B
CASpecie A
SNP 2SNP 1Specie
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PROTOCOLLO SPERIMENTALE
SpecieA 95 AGTCAAYCGRCCGTTCGCCCGCTGACTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieB 95 AGTCAAYCGACCGTTCCACCGCTGAGTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieC 95 AGTCAAYCGGCCGTTCGACCGTTGAGTATACGGCGTCAGTAGGCACTYAYCCGACTRAAT
SpecieA 155 ACTTYCGGGCAATCTACGRYYTCAAYCGRCAGTTCGGRTCACGACGACGTCYTRCAGTTC
SpecieB 155 ACYTYCGRGCAATCAACGRYCTCAAYCGRCAGTYCGGRTCACGACGACGTCYTRCAGTTC
SpecieC 155 ACTTYCGGGCAATCGACGRYYTCAATCGRCCGTTCGGATCACGACTACGTCYTRCAGTTC
SpecieA 215 TCTACGTACAGTCACGACCTCGCTGAGTATTCGTCYTRCAGMCCGTTCCACCGCACGACG
SpecieB 215 TCTACGTACAGTCACGACTTCGCTGAGTATGCGTCYTRCAGACCGGTCCACCGCRCGACG
SpecieC 215 TCTACGTACAGTCACGACTTCGCTGAGTATACGTCYTRCAGACCGGTCCACCGCACGACG
TGSpecie C
TASpecie B
CASpecie A
SNP 2SNP 1Specie
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PROTOCOLLO SPERIMENTALE
SpecieA 95 AGTCAAYCGRCCGTTCGCCCGCTGACTATACGACGTCAGTAGGCACTYAYCCGACTRAAT
SpecieB 95 AGTCAAYCGACCGTTCCACCGCTGAGTATACGACGTCAGTAGGCACTYAYCCGACTRAYT
SpecieC 95 AGTCAAYCGGCCGTTCGACCGTTGAGTATACGGCGTCAGTAGGCACTYAYCCGACTRAAT
SpecieA 215 TCTACGTTCAGTCACGACGCCGCTGAGTATACGTCYTRCAGMCCGTTCCACCGCACGACG
SpecieB 215 TCTACGTTCAGTCACGACGTCGCTGAGTATCCGTCYTRCAGACCGGTCCACCGCRCGACG
SpecieC 215 TCTACGTTCAGTCACGACGTCGCTGAGTATTCGTCYTRCAGACCGGTCCACCGCACGACG
5’-(T)14CGTTCGCCCGCTGACTATACG-3’ PRIMER 1 (35 basi)
5’-(T)5TACGTTCAGTCACGACG-3’ PRIMER 2 (22 basi)
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PROTOCOLLO SPERIMENTALE
4. REAZIONE SNuPE (MULTIPLEX)
PRODOTTO di PCR PURIFICATO
READY REACTION MIX
AmpliTaq®DNA Polymerase;FS Reaction Buffer;
ddNTPs (ddGTP,ddCTP,ddUTP,ddATP)
PRIMER 1 PRIMER 2
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PROTOCOLLO SPERIMENTALE
4. REZIONE SNuPE (MULTIPLEX)
DenaturazioneDenaturazione..
A
AnnealingAnnealing del primer.del primer.
A5’ 3’
T 5’3’
C
G
5’ 3’T 5’3’
CGSpecie ASpecie A
Specie ASpecie A
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PROTOCOLLO SPERIMENTALE
4. REAZIONE SNuPE (MULTIPLEX)
Estensione del primer.Estensione del primer.
AA
T
C 3’5’
5’3’ GCSpecie ASpecie A
I
lunghezza
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PROTOCOLLO SPERIMENTALE
5. ELETTROFORESI CAPILLARE (MULTIPLEX)
Elettroforesi capillare.Elettroforesi capillare.A5’ 3’
T 5’3’
C
G
A5’ 3’
T 5’3’
T
A
Specie CSpecie C
Specie BSpecie B
G5’ 3’
C 5’3’
T
A
A
A
G
C
T
T
I
lunghezza
I
lunghezzaI
lunghezza
Specie ASpecie A
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PROTOCOLLO SPERIMENTALE
5. ELETTROFORESI CAPILLARE (MULTIPLEX)
Elettroforesi capillare.Elettroforesi capillare.
I
lunghezza
A
G
C
T
A5’ 3’
T 5’3’
C
GSpecie ASpecie A
Specie CSpecie CG5’ 3’
C 5’3’
T
A
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PROTOCOLLO SPERIMENTALE
5. ELETTROFORESI CAPILLARE (MULTIPLEX)
Elettroforesi capillare.Elettroforesi capillare.
AT
A C
Specie BSpecie B
Specie ASpecie A
3’5’
5’3’ GC
I
lunghezzaAT
A T 3’5’
5’3’ AT
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VANTAGGI TECNICA SNuPE
Single Nucleotide Primer Extension
RAPIDITA’ di ESECUZIONE
SENSIBILITA’
RILEVAZIONE SNPs in QUALSIASI PUNTO del MARCATORE SCELTO