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dsRNA È¥ÀÔÀÌ Àû¾î ÀÓ»óºÐ¾ßÀÇ ¿¬±¸°³¹ß¿¡ ÀûÇÕÇÑ

PrimeCap¢â T7 RNA Polymerase (low dsRNA)

-

Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Takara
2560A
PrimeCap¢â T7 RNA Polymerase (low dsRNA)
°ü·ÃÇмú ±¸¸ÅÇϱâ
20,000 U
333,000¿ø  2560A_DS.v2401Da.pdf

Á¦Ç°¼³¸í
PrimeCap¢â T7 RNA Polymerase (low dsRNA)´Â cap analog¸¦ »ç¿ëÇÑ RNA ÇÕ¼ºÀÌ °¡´ÉÇÏ°í, dsRNA (double-strand RNA)ÀÇ »ý¼ºÀ²ÀÌ ³·À¸¸ç, ³»¿­¼ºÀ» (~52¡É) °¡Áöµµ·Ï °³·®µÈ T7 RNA Polymerase*1ÀÌ´Ù. ÀÌ È¿¼Ò´Â in vitro transcription (IVT) °úÁ¤¿¡¼­ CleanCap¢ç Reagent AG (TriLink)µîÀÇ cap analog¿Í °°ÀÌ »ç¿ëÇØ °íÈ¿À² capping °ú °í¼öÀ²ÀÇ mRNA ÇÕ¼ºÀ» ´Þ¼ºÇÏ´Â µ¿½Ã¿¡ dsRNAÀÇ È¥ÀÔÀ¸·Î ÀÎÇØ ¹ß»ýÇÏ´Â ¸é¿ª¿ø¼ºÀ» Å©°Ô ÁÙÀÏ ¼ö ÀÖ´Ù. À̸¦ ÅëÇØ °íÇ°ÁúÀÇ mRNA¸¦ ´ë·®À¸·Î Á¦Á¶ÇÒ ¼ö Àֱ⠶§¹®¿¡ ¹é½Å°ú °°Àº RNA therapy ºÐ¾ßÀÇ ¿¬±¸ °³¹ß¿¡ ÀûÇÕÇÏ´Ù.

*1 T7 promoter ¼­¿­À» ÁÖÇüÀ¸·Î, NTP¸¦ ±âÁú·Î »ç¿ëÇØ double-stranded DNA¿¡¼­ promoterÀÇ downstream ¿µ¿ªÀ» RNA·Î ÇÕ¼º (transcription)ÇÏ´Â È¿¼Ò.

ÀϹÝÀûÀÎ T7 promoter ¼­¿­ ¹× transcription start site (CleanCap Reagent AG »ç¿ë ½Ã ÁÖÀÇ»çÇ×)
IVT¸¦ À§ÇØ ÇÊ¿äÇÑ template DNAÀÇ ÀϹÝÀûÀÎ T7 ÇÁ·Î¸ðÅÍ ¼­¿­Àº ¾Æ·¡¿Í °°´Ù. À̶§ ÇÕ¼ºÇÏ°íÀÚ ÇÏ´Â RNAÀÇ ¿°±â ¼­¿­ÀÇ Æ¯Â¡°ú cap analog »ç¿ë ¿©ºÎ¿¡ µû¶ó TemplateÀ» ´Ù¸£°Ô ¼³°èÇØ¾ß ÇÑ´Ù.

[CleanCap Reagent AG »ç¿ë½Ã]
Transcription start site (+1)ÀÇ NGG¼­¿­À» AGG·Î ¼³°èÇÏ´Â °ÍÀ» ±ÇÀå

[uncapped RNA¸¦ ÇÕ¼ºÇϰųª ARCA¸¦ »ç¿ëÇÏ´Â °æ¿ì]
Transcription start site (+1)ÀÇ NGG¼­¿­À» GGG·Î ¼³°èÇÏ´Â °ÍÀ» ±ÇÀå

±×¸² 1. FLuc mRNA (¾à 1.9 kb)ÀÇ ÇÕ¼º ¼öÀ² (¿ÞÂÊ) ¹× dsRNA »ý¼ºÀ² (¿À¸¥ÂÊ).
Positive Control Template (FLuc) (Takara IVTpro¢â T7 mRNA Synthesis Kit (Code 6144)ÀÇ ±¸¼ºÇ°)°ú T7 RNA Polymerase ver.2.0 (Code 2541A)À¸·Î mRNA ÇÕ¼º ½Ã (20 §¡, »ç¿ë¿¹ Âü°í), mRNA ÇÕ¼º ¼öÀ²ÀÇ ÀúÇÏ ¾øÀÌ dsRNA »ý¼ºÀ²À» 10% ÀÌÇÏ·Î ÁÙ¾îµå´Â °ÍÀ» È®ÀÎÇÒ ¼ö ÀÖ´Ù.


±×¸² 2. IVT ¹ÝÀÀ½Ã »ý¼ºµÇ´Â dsRNAÀÇ ¿¹
º¸Á¸: -20 ¡É
³»¿ë

±¸¼ºÇ°

¿ë·®

PrimeCap¢â T7 RNA Polymerase (low dsRNA)

20,000 U

10X T7 RNA Polymerase Buffer

1 §¢

³óµµ: 200 U/§¡
¿ëµµ
- Cap analog¸¦ »ç¿ëÇÑ capped mRNA ÇÕ¼º (co-transcriptional capping)
- Capping enzymeÀ» Àû¿ëÇϱâ À§ÇÑ uncapped RNA ÇÕ¼º (Âü°í; cap analog ´ëºñ 10~40% ³·Àº RNA ¼öÀ²)
±â¿ø
Escherichia coli carrying a plasmid containing the gene for phage T7 RNA polymerase variant
ÀϹÝÀûÀÎ ¼ºÁú
- ºÐÀÚ·®: ¾à 99.8 kDa
- Cofactor: Mg2+
È°¼ºÀÇ Á¤ÀÇ
37 ¡É¿¡¼­ 1½Ã°£ µ¿¾È 1nmol [3H] GMP¸¦ acid-insoluble fraction¿¡ »ý¼ºÇÏ´Â È¿¼ÒÀÇ ¾çÀ» 1 UÀ¸·Î ÇÑ´Ù.
È°¼º ÃøÁ¤¿ë ¹ÝÀÀ¾×ÀÇ Á¶¼º

40 mM

 

Tris-HCl, pH 8.0

8 mM

 

MgCl2

2 mM

 

spermidine)

5 mM

 

DTT

0.4 mM

 

ATP, UTP, and CTP

0.4 mM

 

[3H]GTP

1 §¶/50 §¡

 

pT7-2 DNA

ÁÖÀÇ»çÇ×
1. º» È¿¼Ò´Â °úµµÇÑ vortexingÀ» ÇÇÇϽñ⠹ٶø´Ï´Ù.
2. Template DNA³ª ¹ÝÀÀ¿¡ »ç¿ëµÇ´Â tube, micro-pipette µîÀÌ RNase¿¡ ¿À¿°µÇ¾î ÀÖ´Â °æ¿ì RNAÀÇ ¼öÀ²ÀÌ ³·¾ÆÁö°Å³ª ´ÜÆíÀÌ ºÐÇØµÉ ¼ö ÀÖ´Ù. µû¶ó¼­ RNase free tube ¹× micro-pipetteÀ» »ç¿ëÇÏ°í ÀÏȸ¿ë Àå°©À» »ç¿ëÇÏ´Â °ÍÀ» ±ÇÀåÇÑ´Ù.
3. ¼±ÇüÈ­µÈ plasmid ȤÀº PCR »ê¹°°°Àº linearized dsDNA¸¦ »ç¿ëÇØ¾ß ÀÏÁ¤ÇÑ ±æÀÌÀÇ RNA¸¦ ÇÕ¼ºÇÒ ¼ö ÀÖ´Ù. À̶§ ¿øÄ¡ ¾ÊÀº ÇüÅÂÀÇ RNA ÇÕ¼ºÀ» ¹æÁöÇϱâ À§ÇØ template DNAÀÇ 3¡¯ ¸»´ÜÀº 5¡¯-protruding ȤÀº blunt ÇüŸ¦ ÃßõÇÑ´Ù.
4. 10X T7 RNA Polymerase Buffer´Â spermidineÀÌ Æ÷ÇԵǾî ÀÖ´Ù. ÀÌ´Â ÇÙ»ê°ú º¹ÇÕü¸¦ Çü¼ºÇØ °æ¿ì¿¡ µû¶ó ºÒ¿ë¼ºÀ¸·Î ħÀüµÇ´Â °æ¿ì°¡ ÀÖ´Ù. µû¶ó¼­ template DNA´Â ¹ÝÀÀ¾× Á¶Á¦ ½Ã enzymeÀ» ³Ö±â ¹Ù·Î Á÷Àü¿¡ ¸¶Áö¸· ¼ø¼­·Î ³Ö´Â °ÍÀ» ±ÇÀåÇÑ´Ù.
5. ÀÌ Á¦Ç°¿¡´Â cap analog ¹× NTP (ATP, CTP, GTP, UTP)°¡ Æ÷ÇԵǾî ÀÖÁö ¾Ê´Ù. µû¶ó¼­ CleanCap¢ç Reagent AG (TriLink)¿Í Ribonucleoside 5£§-Triphosphates (Code 4041 ¿Ü) µîÀÌ º°µµ·Î ÇÊ¿äÇÏ´Ù.
6. IVT½Ã RNA ÇÕ¼º ¹ÝÀÀÀ» ÃËÁøÇÏ´Â Pyrophosphatase (inorganic) (Code 2450A)¿Í ÇÕ¼ºµÈ RNAÀÇ ºÐÇظ¦ ¾ïÁ¦ÇÏ´Â Recombinant RNase Inhibitor ver.2.0 (Code 2315A)¸¦ °°ÀÌ »ç¿ëÇÏ´Â °ÍÀ» ±ÇÀåÇÑ´Ù. ÀÚ¼¼ÇÑ ³»¿ëÀº °¢ Á¦Ç°ÆäÀÌÁö¸¦ Âü°í ¹Ù¶ø´Ï´Ù.
»ç¿ë ¿¹

RNase-free Water

 

X §¡

10X T7 RNA Polymerase Buffer

 

2 §¡

ATP, CTP, GTP, UTP*1

 

°¢ 10 mM

CleanCap Reagent AG*2

 

4 mM

Template DNA

 

0.5 ~ 2 §¶

Recombinant RNase Inhibitor ver.2.0

 

20 U

Pyrophosphatase (inorganic)

 

0.1 U

PrimeCap T7 RNA Polymerase

 

200 U

Total

 

20 §¡


37¡É¿¡¼­ 1~2½Ã°£ ¹ÝÀÀ
*1 Modified NTP¸¦ »ç¿ëÇÏ´Â °æ¿ì ÇØ´çÇÏ´Â NTP¸¦ µ¿·®À¸·Î ´ëüÇÏ¿© »ç¿ë
*2 CleanCap Reagent AG (TriLink BioTechnologies: Code. N-7113-1/5/10)
°ü·ÃÁ¦Ç°
- IVT¿ë Plasmid templateÀ» °£´ÜÇÏ°Ô Á¦ÀÛÇÏ´Â Kit: Cloning Kit for mRNA Template (Code 6143)
- Capping enzyme (uncapped RNA -> cap-0 RNA): Vaccinia Capping Enzyme (Code 2460A)
- Capping enzyme (cap-0 RNA -> cap-1 RNA): mRNA Cap 2-O-Methyltransferase (Code 2470A)
- IVTÀü vectorÀÇ ¼±ÇüÈ­¿¡ »ç¿ëÇÏ´Â Type IIS Á¦ÇÑÈ¿¼Ò: BspQ I (Code 1227A)
- BspQ I Àνļ­¿­ÀÌ ÀÖ´Â IVT template vector: Template Vector (BspQ I) for T7 mRNA Synthesis (Code 6146)
- mRNA ¹ßÇöÀ» Boost½Ã¾àÀÌ Æ÷ÇÔµÈ transfection reagent: TransIT¢ç-mRNA Transfection Kit (Code MIR 2250)

Keyword :

¼ö½ÄÈ¿¼Ò£¯DNA Polymerase¡¢RNA Polymerase
>
dsRNA È¥ÀÔÀÌ Àû¾î ÀÓ»óºÐ¾ßÀÇ ¿¬±¸°³¹ß¿¡ ÀûÇÕ..
PrimeCap¢â T7 RNA Polymerase (low dsRNA)