[PrimeSTAR ´Â]
´ÙÄ«¶ó°¡ µ¶ÀÚÀûÀ¸·Î °³¹ßÇÑ ¸Å¿ì ³ôÀº Á¤È®¼º°ú ¶Ù¾î³ ÁõÆøÈ¿À²À» °âºñÇÑ PCR È¿¼ÒÀÔ´Ï´Ù.
¸Å¿ì °·ÂÇÑ 3' ¡æ 5' exonuclease È°¼ºÀ» °¡Á®, PCR ÁõÆø¿¡ ´ëÇØ °·ÂÇÑ ±³Á¤±â´É (Proofreading ±â´É)À» ³ªÅ¸³»´Â ÇÑÆí,
Taq DNA Polymerase ¸¸Å ¸Å¿ì ¶Ù¾î³ ÁõÆø È¿À²µµ ³ªÅ¸³À´Ï´Ù.
»ó¿Â¿¡¼ÀÇ DNA polymerase È°¼º ¹× 3' ¡æ 5' exonuclease È°¼ºÀ» ¾ïÁ¦Çϱâ À§ÇÑ monoclonal antibody¸¦ Æ÷ÇÔÇÏ°í ÀÖ¾î PCR ¹ÝÀÀÀüÀÇ mispriming À̳ª primer digestionÀ» ¸·°í ³ôÀº ¹ÝÀÀƯÀ̼ºÀ» ¾òÀ» ¼ö ÀÖ½À´Ï´Ù.
[Á¤È®ÇÑ PCR, CloningÀ» À§ÇÑ ÃÖ»óÀÇ ¶óÀξ÷] - PrimeSTAR
¢ç DNA Polymerase ½Ã¸®Á Ư¡Àº ¾Æ·¡¿Í °°½À´Ï´Ù.
|
Á¤È®µµ |
GC or AT rich
¼¿ÀÇ ÁõÆø |
ÁÖÇü·®
Àû¿ë¹üÀ§ |
½ÅÀå¼Óµµ |
ÁõÆø±æÀÌ
(human gDNA±âÁØ) |
PCR »ê¹° ¸»´ÜÇü»ó |
Hot Start |
PrimeSTAR¢ç GXL |
¡Ú¡Ú¡Ú¡Ú |
¡Ú¡Ú¡Ú¡Ú¡Ú |
¡Ú¡Ú¡Ú¡Ú¡Ú |
¡Ú¡Ú¡Ú¡Ú(¡Ú) * |
30 kb |
Blunt end |
Anti-Taq antibody·Î º°µµÀÇ activation °úÁ¤¾øÀÌ hot start PCR °¡´É |
PrimeSTAR¢ç Max |
¡Ú¡Ú¡Ú¡Ú¡Ú |
¡Ú¡Ú¡Ú |
¡Ú¡Ú¡Ú(¡Ú)** |
¡Ú¡Ú¡Ú¡Ú¡Ú |
6 kb |
PrimeSTAR¢ç HS |
¡Ú¡Ú¡Ú¡Ú¡Ú |
¡Ú¡Ú¡Ú |
¡Ú¡Ú |
¡Ú¡Ú |
8.5 kb |
* È¿¼Ò 2¹è »ç¿ë½Ã 10ÃÊ/kb °í¼Ó PCR °¡´É
** ½ÅÀå½Ã°£ 1ºÐ/kb ¼³Á¤½Ã °¡´É
[PrimeSTAR ½Ã¸®Áî º° Á¦Ç°º¸±â] - ½Ã¸®Áî º°·Î È¿¼Ò ´ÜÇ°, premix µî ´Ù¾çÇÑ ÇüÅÂÀÇ Á¦Ç°À» ¼±ÅÃÇÒ ¼ö ÀÖ½À´Ï´Ù.
|
Code |
Á¦Ç°¸í |
¿ë·® |
PrimeSTAR¢ç GXL |
R050A |
PrimeSTAR¢ç GXL DNA Polymerase |
250 U |
R051A |
PrimeSTAR¢ç GXL Premix |
200 ȸ* |
R052A |
PrimeSTAR¢ç GXL Premix Fast, Dye plus |
200 ȸ* |
PrimeSTAR¢ç Max |
R045A |
PrimeSTAR¢ç Max DNA Polymerase |
100 ȸ* |
PrimeSTAR¢ç HS |
R010A |
PrimeSTAR¢ç HS DNA Polymerase |
250 U |
R040A |
PrimeSTAR¢ç HS (Premix) |
100 ȸ* |
R044A |
PrimeSTAR¢ç HS DNA Polymerase with GC Buffer |
250 U |
*50 §¡ PCR ¹ÝÀÀ ½Ã
[PrimeSTAR ¼±Åð¡À̵å]
PrimeSTAR¢ç Max DNA Polymerase Ư¡º¸±â
1. Á¤È®¼º
GC richÀÎ
Thermus thermophilus HB8 genomic DNAÀ» ÁÖÇüÀ¸·Î ÀÓÀÇ·Î ¼±ÅÃÇÑ 8¿µ¿ª (ÁõÆø »çÀÌÁî´Â °¢°¢ ¾à 500 bp)À» PCR ÁõÆøÇÑ ÈÄ, º¤ÅÍ¿¡ cloning ÇÏ¿©
°¢ ÁõÆø»ê¹°¸¶´Ù ´Ù¼öÀÇ cloneÀ» ¼±º°ÇÏ¿© sequencingÀ» ÁøÇàÇÏ¿´´Ù. ±× °á°ú PrimeSTAR
¢ç Max DNA Polymerase´Â ÃÑ 230,129 ¿°±â Áß ´Ü 9°³ÀÇ ¿°±â¿¡¼¸¸
mutationÀÌ ³ªÅ¸³²À» È®ÀÎÇÏ¿´´Ù. ÀÌ´Â
Taq DNA Polymerase°ú ºñ±³Çؼ ¾à 10¹è ³ôÀº Á¤È®µµÀ̸ç, PrimeSTAR
¢ç HS DNA Polymerase¿Í A»ç High Fidelity È¿¼Ò¿¡ ºñÇؼµµ
µ¿µî ÀÌ»óÀÇ Á¤È®¼ºÀ» º¸¿´´Ù.
[°¢ È¿¼ÒÀÇ Á¤È®µµ ºñ±³]
2. ÁõÆø¼Óµµ
1)
¥ë DNAÀ» ÁÖÇüÀ¸·Î annealing ½Ã°£ 5ÃÊ, extension ½Ã°£ 10ÃÊ È¤Àº 30ÃÊ·Î °¢ »çÀÌÁ ÁõÆøÇÏ¿´´Ù.
Template |
¥ë DNA 1 ng/50§¡ ¹ÝÀÀ°è |
Thermal cycler |
TaKaRa PCR Thermal Cycler Dice¢ç |
PCR Á¶°Ç |
98¡É
55¡É
72¡É |
10 sec.
5 sec.
10 or 30 sec. |
30 cycles |
[½ÅÀå ½Ã°£ 10ÃÊ] |
[½ÅÀå ½Ã°£ 30ÃÊ] |
|
M: ¥ë-Hind III digest |
Extension ½Ã°£ 10ÃÊ ¼³Á¤½Ã 6 kbp, 30ÃÊ ¼³Á¤½Ã 8 kb±îÁö ¾çÈ£ÇÑ ÁõÆøÀ» º¼ ¼ö ÀÖ°í,
¥ë DNAÀ» ÁÖÇüÀ¸·Î PCR ÇÏ´Â °æ¿ì extension ½Ã°£ 5 sec./kbÀÇ
¼³Á¤ÀÌ °¡´ÉÇÑ °ÍÀ» È®ÀÎÇÏ¿´´Ù.
2) Human genomic DNA¸¦ ÁÖÇüÀ¸·Î annealing ½Ã°£ 5ÃÊ, extension ½Ã°£ 10ÃÊ È¤Àº 30ÃÊ·Î °¢ »çÀÌÁ ÁõÆøÇÏ¿´´Ù.
Template |
Human gDNA 100 ng/50§¡ ¹ÝÀÀ°è |
Thermal cycler |
TaKaRa PCR Thermal Cycler Dice¢ç |
PCR Á¶°Ç |
98¡É
55¡É
72¡É |
10 sec.
5 sec.
10 or 30 sec. |
30 cycles |
[½ÅÀå ½Ã°£ 10ÃÊ] |
[½ÅÀå ½Ã°£ 30ÃÊ] |
|
M: ¥ë-Hind III digest |
Extension ½Ã°£ 10ÃÊ ¼³Á¤½Ã 4 kb, 30ÃÊ ¼³Á¤½Ã 6 kb±îÁö ¾çÈ£ÇÑ ÁõÆøÀ» º¼ ¼ö ÀÖ°í, human gDNA¸¦ ÁÖÇüÀ¸·Î PCR ÇÏ´Â °æ¿ì¿¡µµ extension ½Ã°£ 5 sec./kbÀÇ
¼³Á¤ÀÌ °¡´ÉÇÑ °ÍÀ» È®ÀÎÇÏ¿´´Ù.
3) cDNA¸¦ ÁÖÇüÀ¸·Î annealing ½Ã°£ 5ÃÊ, extension ½Ã°£ 10ÃÊ È¤Àº 30ÃÊ·Î °¢ »çÀÌÁ ÁõÆøÇÏ¿´´Ù.
Template |
cDNA (Total RNA 200 ng)/50§¡ ¹ÝÀÀ°è |
Thermal cycler |
TaKaRa PCR Thermal Cycler Dice¢ç |
PCR Á¶°Ç |
98¡É
55¡É
72¡É |
10 sec.
15 sec.
10 or 30 sec. |
30 cycles |
[½ÅÀå ½Ã°£ 10ÃÊ] |
[½ÅÀå ½Ã°£ 30ÃÊ] |
M: ¥ë-Hind III digest |
Extension ½Ã°£ 10ÃÊ ¼³Á¤½Ã 2 kb, 30ÃÊ ¼³Á¤½Ã 4 kb±îÁö ¾çÈ£ÇÑ ÁõÆøÀ» º¼ ¼ö ÀÖ°í, cDNA¸¦ ÁÖÇüÀ¸·Î PCR ÇÏ´Â °æ¿ì¿¡µµ extension ½Ã°£ 5~10 sec/kbÀÇ ¼³Á¤ÀÌ ÇÊ¿äÇÑ °ÍÀ» È®ÀÎÇÏ¿´´Ù.
PrimeSTAR¢ç GXL DNA Polymerase Ư¡º¸±â
1. ½ÅÀ强
¶Ù¾î³ ½ÅÀ强À» °¡Áø PrimeSTAR
¢ç GXLÀº ±âÁ¸ÀÇ High Fidelity PCRÈ¿¼Ò°¡ ÁõÆøÇÏÁö ¸øÇÑ ±ä Ÿ°Ùµµ ÁõÆøÇÒ ¼ö ÀÖ´Ù.
Human genomic DNA¸¦ ÁÖÇüÀ¸·Î 30 kb,
¥ë DNA¸¦ ÁÖÇüÀ¸·Î 40 kb, cDNA¿¡¼´Â 13.5 kb±îÁö ÁõÆøµÇ´Â °ÍÀ» È®ÀÎÇÏ¿´´Ù.
1. |
p53 |
0.5 kb |
2. |
DCLRE 1A |
1 kb |
3. |
DCLRE 1A |
2 kb |
4. |
DCLRE 1A |
4 kb |
5. |
¥â-globin |
8.5 kb |
6. |
¥â-globin |
15 kb |
7. |
¥â-globin |
20 kb |
8. |
¥â-globin |
24 kb |
9. |
¥â-globin |
27 kb |
10. |
¥â-globin |
30 kb |
M. |
¥ë-Hind III digest |
1. |
0.5 kb |
2. |
1 kb |
3. |
2 kb |
4. |
4 kb |
5. |
8 kb |
6. |
15 kb |
7. |
20 kb |
8. |
30 kb |
9. |
40 kb |
M. |
¥ë-Hind III digest |
1. |
Dystrophin |
1 kb |
2. |
Dystrophin |
2 kb |
3. |
CCND2 |
2.8 kb |
4. |
TFR |
4 kb |
5. |
Dystrophin |
6 kb |
6. |
Dystrophin |
8 kb |
7. |
Dystrophin |
12 kb |
8. |
Dystrophin |
13.5 kb |
M. |
¥ë-Hind III digest |
2. ³ôÀº °¨µµ¿Í ³ÐÀº ÁÖÇü Çã¿ë·®
ÀϹÝÀûÀ¸·Î Á¤È®¼ºÀÌ ³ôÀº high fidelity PCR È¿¼Ò´Â ¹ÝÀÀ¾×¿¡ Æ÷ÇÔµÈ template ¾ç¿¡ ¿µÇâÀ» ¹Þ±â ½±°í, cDNAÀÇ ÁõÆøÀÌ ±î´Ù·Î¿î °æ¿ì°¡ ÀÖ´Ù. ÇÏÁö¸¸ PrimeSTAR
¢ç
GXLÀº template ¾ç¿¡ ´ëÇÑ Çã¿ë ¹üÀ§°¡ ³Ð°í cDNA¸¦ ÁÖÇüÀ¸·Î ÇÑ ÁõÆøµµ È¿À²ÀûÀ¸·Î ÁøÇàÇÒ ¼ö ÀÖ´Ù.
´Ù¾çÇÑ ¾çÀÇ human genomic DNA¸¦ ÁÖÇüÀ¸·Î PrimeSTAR GXL
¢ç °ú Ÿ»ç High Fidelity PCR È¿¼Ò ¹× r
Taq ÀÇ ÁõÆø È¿À²À» ºñ±³ÇÏ¿´´Ù.
ÁÖÇü: human genomic DNA
Ÿ°Ù: DCLRE 1A À¯ÀüÀÚ (2 kb)
Template ¾ç /50 §¡ ¹ÝÀÀ°è |
1. |
No template |
2. |
100 pg |
3. |
1 ng |
4. |
10 ng |
5. |
100 ng |
6. |
200 ng |
7. |
500 ng |
M. |
¥ë-Hind III digest |
PrimeSTAR
¢ç GXLÀº Ÿ»ç High Fidelity PCRÈ¿¼Ò³ª r
Taqº¸´Ù ¹Î°¨ÇÏ°í ¾çÈ£ÇÑ ÁõÆø¼ºÀ» º¸¿´´Ù. ¶Ç, Ÿ»ç High Fidelity PCR È¿¼Ò´Â ¸¹Àº ¾çÀÇ templateÀÌ Æ÷ÇÔµÈ Á¶°Ç¿¡¼
ÁõÆø ¾ïÁ¦¹ÝÀÀÀÌ ³ªÅ¸³µÁö¸¸, PrimeSTAR GXL
¢ç´Â template ¾ç¿¡ °ü°è¾øÀÌ ³ôÀº ¹ÝÀÀ¼ºÀ» ³ªÅ¸³»¾ú´Ù.
3. GC rich Ÿ°ÙÀÇ ÁõÆø
ºñƯÀÌÀû ÁõÆøÀÌ ÀϾ±â ½¬¿î GC rich template¿¡¼µµ Ưº°ÇÑ ¹öÆÛÀÇ »ç¿ëÀ̳ª ¹ÝÀÀ Á¶°ÇÀ» º¯°æÇÏÁö ¾Ê¾Æµµ ƯÀ̼ºÀÌ ³ôÀº ÁõÆøÀÌ °¡´ÉÇÏ´Ù.
(ÀÚ»ç ºñ±³ µ¥ÀÌÅÍ)
ÁÖÇü |
Ÿ°Ù À¯ÀüÀÚ |
Human genomic DNA
(100 ng/50 §¡ ¹ÝÀÀ°è) |
1: APOE gene 746 bp (GCÇÔ·® 74%) |
2: TGF¥â 1 gene 2,005 bp (GCÇÔ·® 69%) |
T. thermophilus HB8 genomic DNA
(10 ng/50 §¡ ¹ÝÀÀ°è) |
3: 2,029 bp (GCÇÔ·® 74%) |
4: 4,988 bp (GCÇÔ·® 74%) |
M: pHY Marker |
B»ç È¿¼Ò 2: GC rich ¿ë È¿¼Ò |
D»ç È¿¼Ò: GC buffer »ç¿ë |