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Home > Learning center > PCR > ´Ù¾çÇÑ ½ÇÇè Àû¿ë »ç·Ê > »ç¶÷ ¼ÕÅé DNA¸¦ ÀÌ¿ëÇÑ ¸ñÀû À¯ÀüÀÚ ÁõÆø

»ç¶÷ ¼ÕÅé DNA¸¦ ÀÌ¿ëÇÑ ¸ñÀû À¯ÀüÀÚ ÁõÆø

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»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

Lane No.

Target gene

Amplicon size

% GC content

ÁõÆø°á°ú

Terra PCR Direct

A»ç È¿¼Ò

B»ç È¿¼Ò

1

UCRchr9*

1.1 kb

26%

+

+

2

EGFR

1.0 kb

50%

+

+

3

TGFβ1

1.0 kb

72%

+

4

UCRchr11**

2.2 kb

27%

+

+

+

5

IGF2R

2.0 kb

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

 

98°C 10 sec

40 cycles

68°C 1 min/kb