Molecular Beacons TaqMan , ( ) (.8.). , " " (stem-loop), .
.8. Molecular Beacons
, , .
2- (LightCycler assay)
Light Cycler , . , 3' , , 5' , , 1-3 (.9.).
Molecular beacons LightCycler , , - (, SNP).
.9. LightCycler assay
SYBR Green C(T) = 35-37, . , real-time PCR.
TaqMan , PCR-, , . , , .
SYBR Green TagMan
SYBR Green . . , TaqMan.
:
1. -: 50-200 .
2. : 58-65OC.
3. : 20-30 .
4. GC : ~40-60%.
5. : .
6. : .
7. G/C 3' ( ).
TaqMan
:
1. GC : 20-80%.
2. : 68-70OC.
3. ( G: 4- ).
4. , C, G.
5. 5'- G.
6. : FAM VIC.
7. : - TAMRA, BHQ1 (Blackhole).
|
|
|
:
1. -: 50 .
2. : 58-60OC.
3. : 20-30 .
4. GC : 20-80%.
5. : .
6. : .
G/C 3' ( ).
2.
SYBR Green TagMan
| SYBR Green | , .. . | , PCR- . - , , . . |
| TaqMan | . : PCR-. ó . | . ( $200) . ( , , , ). |
. :
1. .
2. 2l 30l.
3. (, , ).
- ( )
:
-
: iQ iCycler, CFX96 Touch Deep Well Real-time PCR Detection System (Bio-Rad), ABI Prism (Applied Biosystems), LightCycler (Roche), SmartCycler (Cepheid). CFX96 "Bio-Rad" (.10).
(Nanodrop ND-1000 )
II
: 2-20 , 10-100 , 20-200 , 200-1000
1,5
-
1,5 25-1000
: 1,5 , 0,5 , 0,2
0,2 .
, 2 80
|
|
|
, -20 0
.10. CFX96 "Bio-Rad" ( - )
:
0,5 , 0,2
0,2
-
.
1. .
2.
3. .
- - 300 nM ;
- ( TaqMan) - 250 nM;
- MgCl2 - 5 nM (SYBR Green), 3.5 nM (TaqMan);
- - AmpliTaqGold, Applied Biosystems.
3. SYBR Green
| 8 | 94 OC | |
| 30 | 94 OC | |
| 20 | 60-65 OC | |
| 1 | 72 OC | |
| Plate read ( ) | ||
| Go to 2 for 39 times ( 2 39) | ||
| Melting curve from 60OC to 94OC read every 0.2OC hold for 1 sec before plate read ( 60 OC 94 OC 0,2 OC 1 Plate read) | ||
| Forever 10 OC ( 10 OC) |
.
1. , , . , 15-20 , .
2. (Plate read) 72OC, ( . ).
3. SYBR Green, , C(T) 35-38, 40 .
4. AmpliTaqGold 60OC, . 3 4 1 min & 60-65OC, .
5. (melting curve) SYBR Green PCR. , PCR- .
4.
