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0°ÇÀÇ Èı⺸±âÀçÁ¶ÇÕ DNA¿Í ÇüÁúÀüȯ¿¡ ´ëÇØ ±â¼úÇÑ ¸®Æ÷Æ® Âü°íÀÚ·áÀÔ´Ï´Ù.
ÀçÁ¶ÇÕ dna (recombinant dna) ÀçÁ¶ÇÕ dna recombinant dna hybrid dna chemeric dna (chimera) ÇüÁúÀüȯ (transformation)
ÇüÁúÀüȯ, Á¢ÇÕ (conjugation), ÇüÁúµµÀÔ (transduction)
ÇüÁúÀüȯ : ÀçÁ¶ÇÕ dna¸¦ ¼÷Á¶ ¼¼Æ÷¿¡ µµÀÔ, ¹ßÇö½ÃÅ°´Â °úÁ¤
transformant transgenic animal, transgenic plant, gmo (genetically modified organism) gmc (genetically modified crop) lmo (living modified organism) gem (genetically engineered microorganism)
plasmid vector¸¦ ÀÌ¿ëÇÑ ÀçÁ¶ÇÕ dna ºÐÀÚÀÇ Á¦ÀÛ
for efficient ligation : Á¦ÇÑÈ¿¼ÒÀÇ ¼±ÅÃÀÌ °¡Àå Áß¿ä
dna fragment ¿Í vector¿¡ sticky end »ý¼º
À¯ÀüÀÚ¸¦ Æ÷ÇÔÇÑ dna¿Í ¿î¹Ýü dna¸¦ °°Àº Á¦ÇÑÈ¿¼Ò·Î ó¸®
°°Àº sticky ends¸¦ »ý¼ºÇÏ´Â ´Ù¸¥ Á¦ÇÑÈ¿¼Ò·Î ó¸®
ligation of sticky ends ÀÚ°¡ ¿¬°á (self ligation) ÀÚ°¡ ¿¬°á ¹æÁö (prevention of self-ligation) vectorÀÇ Å»ÀλêÈ (dephosphorylation)
Á¦ÇÑÈ¿¼Ò·Î Àý´ÜµÈ vector dnaÀÇ ÇÑ ÂÊ ¸»´ÜÀ¸·ÎºÎÅÍ 5¡¯-po4 Á¦°Å
ligation : 3¡¯-oh 5¡¯-po4 dephosphorylation of 5¡¯-po4 ¡æ 5¡¯-oh 3¡¯-oh 5¡¯-oh ¡æ no ligation
alkaline phosphatase (cip, calf intestinal phosphatase)
º¤ÅÍÀÇ ÀÚ°¡ ¿¬°áÀ» ¸·±â À§ÇÑ Å»ÀλêÈ È¿¼ÒÀÇ Ã³¸®
ligation of dna fragments with blunt ends blunt end ¡æ sticky end linker (¿¬°áÀÚ) adaptor homopolymer tailing dna topoisomerase¸¦ ÀÌ¿ëÇÑ blunt end ¿¬°á linker
synthetic oligonucleotide (dsdna) : xxx-xxx bp
recognition sites for xxx-xxx restriction enzymes
ligated to blunt end dna by t4 dna ligase efficient ligation at high concentration
generation of cohesive ends : digestion with re
problem : the sites for the enzyme used to generate
cohesive ends may be present in the target dna fragment.
creation of cohesive ends on blunt-ended dna fragments.
suitable linkers are liga
ted to the blunt ends by t4 dna ligase.the linker is then cleaved with the appropriate restriction enzyme to generate sticky ends.
adaptor linker with cohesive ends linker digested with re
one blunt end : 5¡¯-po4 one sticky end : 5¡¯-oh
3¡¯-oh 3¡¯-oh no self-ligation :
t4 dna ligase to ligate adaptors to the blunt-ended dna (ÀÌÇÏ »ý·«)
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Æ÷ÀÎÆ® : ÀÚ·á 1°Ç´ç ÃÖ´ë 5,000P Áö±Þ
Çö±Ý : ÀÚ·á 1°Ç´ç ÃÖ´ë 2,000¿ø Áö±Þ