»ç¶÷ ¼ÕÅé DNA¸¦ ÀÌ¿ëÇÑ ¸ñÀû À¯ÀüÀÚ ÁõÆø
(Amplifying a gene of interest from human nail DNA)
Terra PCR Direct Polymerase¸¦ ÀÌ¿ëÇÏ¿© GC-rich target°ú Å©±â°¡ Å« target (4 kb)ÀÇ direct amplification½Ã Ÿ»çÀÇ ´Ù¸¥ È¿¼Òº¸´Ù ¶Ù¾î³ ¼º´ÉÀ» °¡Áö°í ÀÖÀ½À» È®ÀÎÇÏ¿´´Ù.
Overview : Terra PCR Direct Polymerase Mix´Â ÃÖÀûÈµÈ DNA polymerase¸¦ Æ÷ÇÔÇÏ°í ÀÖ´Ù.
Ÿ»ç PCR È¿¼Ò¿ÍÀÇ ºñ±³: Terra PCR Direct Polymerase´Â GC-rich target ÁõÆø¿¡ Ź¿ùÇÑ ´É·ÂÀ» °¡Áö°í ÀÖÀ½À» È®ÀÎÇÏ¿´´Ù.
¡á Introduction
Terra PCR Direct Polymerase Mix´Â DNA Á¤Á¦ ¾øÀÌ Á¶Á÷À̳ª crude extracts¿¡¼ Á÷Á¢ ÁõÆøÀÌ °¡´ÉÇϵµ·Ï ÃÖÀûÈµÈ DNA polymerase¸¦ Æ÷ÇÔÇÏ°í ÀÖ´Ù. º» È¿¼Ò´Â ÃÖ´ë 4kb±îÁö ÁõÆø °¡´ÉÇϸç, ¶ÇÇÑ ÁõÆøÀÌ ±î´Ù·Î¿î ÁÖÇü (¿¹½Ã - GC ÇÔ·® 70% ÀÌ»ó) ¿ª½Ã ÁõÆøÇÒ ¼ö ÀÖ´Ù. ÀÌ È¿¼Ò´Â PCR ¾ïÁ¦Á¦ (inhibitor), growth serum ¹× ±× ¿Ü template¿Í °°ÀÌ È¥À﵃ ¼ö ÀÖ´Â ¹°ÁúÀÌ ÀÖ´Â »óÅ¿¡¼µµ DNA ÁõÆøÀÌ °¡´ÉÇϱ⠶§¹®ÀÌ´Ù.
¡á Results
Ÿ»ç PCR È¿¼Ò¿ÍÀÇ ºñ±³
Terra PCR Direct Polymerase Mix´Â »ç¶÷ ¼ÕÅé DNA°¡ Æ÷ÇÔµÈ crude extract¿¡¼ GC-rich target°ú ¾à 4kb±îÁöÀÇ targetÀ» Ÿ»çÀÇ È¿¼Òº¸´Ù ¶Ù¾î³ª°Ô ÁõÆøÇÔÀ» È®ÀÎÇÏ¿´´Ù.
Terra PCR Direct Polymerase Mix (Panel A)´Â ¸ðµç »ùÇÿ¡¼ ¼º°øÀûÀ¸·Î ¸ñÀû À¯ÀüÀÚ¸¦ ÇÕ¼ºÇÏ¿´´Ù. A»çÀÇ È¿¼Ò (Panel B)´Â AT-rich sample (Lane 4)¿¡¼ Èñ¹ÌÇÑ ¸ñÀû band¸¦ È®ÀÎÇÏ¿´À¸³ª, ³ª¸ÓÁö »ùÇÿ¡¼´Â ¸ðµÎ ÁõÆøÇÏÁö ¸øÇÏ¿´´Ù. B»ç È¿¼Ò (Panel C)´Â GC-rich target (Lane 3, 6)Àº ÁõÆøÇÏÁö ¸øÇÏ¿´°í, target size°¡ ±æ¾îÁú¼ö·Ï ÁõÆø È¿À²ÀÌ °¨¼ÒÇÏ¿´´Ù (Lanes 2, 5, 8).
[±×¸²] »ç¶÷ ¼ÕÅé DNA°¡ Æ÷ÇÔµÈ crude extract¿¡¼ 3°³ÀÇ DNA polymerase¸¦ ÀÌ¿ëÇÏ¿© ´Ù¾çÇÑ Å©±â¿Í ´Ù¾çÇÑ GC contentÀÇ ¸ñÀûÀ¯ÀüÀÚ¸¦ ÁõÆøÇÏ¿© ±× °á°ú¸¦ agarose gel Àü±â¿µµ¿À¸·Î ºñ±³ÇÏ¿´´Ù. Terra PCR Direct Polymerase Mix´Â GC-rich template´Â ¹°·Ð ÃÖ´ë 4kb ±æÀÌÀÇ ¸ñÀû À¯ÀüÀÚ±îÁö ÁõÆøÇÒ ¼ö ÀÖ´Â ¹Ý¸é (Panel A), A»ç È¿¼Ò´Â AT-rich sample Çϳª¸¦ Á¦¿ÜÇÏ°í ÁõÆø È¿À²ÀÌ ¸Å¿ì ³·°Å³ª ÀüÇô ÁõÆøµÇÁö ¾Ê¾Ò´Ù (Panel B). B»ç È¿¼Ò¸¦ »ç¿ëÇÑ °æ¿ì´Â GC content 50% À̻󿡼´Â ÁõÆøÀÌ µÇÁö ¾Ê¾Ò´Ù (Panel C) (Lane M: pHY marker).
Amplification results for Terra Direct compared to two other DNA polymerases
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Lane No.
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Target gene
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Amplicon size
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% GC content
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ÁõÆø°á°ú
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Terra PCR Direct
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A»ç È¿¼Ò
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B»ç È¿¼Ò
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1
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UCRchr9*
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1.1 kb
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26%
|
+
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–
|
+
|
2
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EGFR
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1.0 kb
|
50%
|
+
|
–
|
+
|
3
|
TGFβ1
|
1.0 kb
|
72%
|
+
|
–
|
–
|
4
|
UCRchr11**
|
2.2 kb
|
27%
|
+
|
+
|
+
|
5
|
IGF2R
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2.0 kb
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50%
|
+
|
–
|
+
|
6
|
IGFβ1
|
2.0 kb
|
69%
|
+
|
–
|
–
|
7
|
FragileX
|
4.0 kb
|
33%
|
+
|
–
|
+
|
8
|
EGFR
|
3.8 kb
|
44%
|
+
|
–
|
–
|
9
|
TGFß1
|
4.0 kb
|
63%
|
+
|
–
|
–
|
*UCRchr9 = Chromosome 9 AT-rich noncoding region.
**UCRchr11 = Chromosome 11 AT-rich noncoding region
¡á Conclusions
ÀÌ ½ÇÇèÀº Terra PCR Direct Polymerase°¡ ¹ýÀÇÇÐ ºÐ¼®¿¡ ÀÚÁÖ »ç¿ëµÇ´Â »ç¶÷ÀÇ ¼ÕÅé°ú °°ÀÌ ¸Å¿ì ´Ü´ÜÇÏ°í, ¿ëÇØÇϱ⠾î·Á¿î Á¶Á÷À» ºÐ¼®ÇÏ´Â µ¥ ÁÁÀº ¼±ÅÃÀÓÀ» º¸¿©ÁÖ¾ú´Ù. Terra PCR Direct Polymerase´Â GC-rich template¿¡¼ ÃÖ´ë 4 kb±îÁö ÁõÆøÇÒ ¼ö ÀÖ¾ú´Ù. ¹Ý¸é, B»ç Polymerase´Â GC content 50% ÀÌ»óÀÎ °æ¿ì À¯ÀüÀÚ¸¦ ÁõÆøÇÏÁö ¸øÇÏ¿´À¸¸ç, A»ç Polymerase´Â AT-rich »ùÇà 1°³¸¦ Á¦¿ÜÇÏ°í´Â ÁõÆøÈ¿À²ÀÌ ¸Å¿ì ³·°Å³ª ÀüÇô ÁõÆøÇÏÁö ¸øÇÏ¿´´Ù.
¡á Methods
5 mg »ç¶÷ ¼ÕÅé »ùÇÃÀ» 44 §¡ SimplePrep™ reagent for DNA (Çѱ¹ÆǸźҰ¡)¿¡ ³Ö¾î, SimplePrep™ reagent for DNAÀÇ ¸Å´º¾ó¿¡ µû¶ó »ç¶÷ ¼ÕÅé DNA¸¦ Æ÷ÇÔÇÏ´Â crude extract¸¦ Á¦ÀÛÇÏ¿´´Ù. Á¦ÀÛÇÑ crude extract¸¦ 2.5 §¡¾¿ ºÐÁÖÇÏ¿©, PCR ¹ÝÀÀ¾× 25 §¡ ±âÁØÀ¸·Î ´Ù¾çÇÑ À¯ÀüÀÚ ÁõÆøÀ» È®ÀÎÇÏ¿´´Ù. °¢ È¿¼ÒÀÇ ¹ÝÀÀ Á¶°ÇÀº °¢ Á¦Á¶»ç¿¡¼ ÃßõÇÏ´Â Á¶°Ç´ë·Î ÀÌ¿ëÇÏ¿´À¸¸ç, ÁõÆø ÈÄ 3 §¡¸¦ agarose gel¿¡ Àü±â¿µµ¿ÇÏ¿© °á°ú¸¦ È®ÀÎÇÏ¿´´Ù.
[Terra PCR Direct Polymerase MixÀÇ PCR ¹ÝÀÀÁ¶°Ç]
98°C 2 min
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98°C 10 sec
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40 cycles
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68°C 1 min/kb
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