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‫ﻫﻨﺪﺳﺔ وراﺛﻴﺔ‬
‫)ورث‪(202‬‬
‫ﺗﺤﻀﻴﺮ الـ‬
‫‪Insert of DNA‬‬
‫اﻟﺠﺰء اﻟﺜﺎﻧﻲ‬ ‫ﻗﺴﻢ اﻟﻮراﺛﺔ – ﻛﻠﻴﺔ اﻟﺰراﻋﺔ – ﺟﺎﻣﻌﺔ‬
‫ﻋﻴﻦ ﺷﻤﺲ‬
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 ‫ﺗﺤﻀﻴﺮ الـ ‪Insert of‬‬ ‫ﻫﻨﺪﺳﺔ وراﺛﻴﺔ‬
‫‪ DNA‬اﻟﺠﺰء اﻟﺜﺎﻧﻲ‬

‫‪3/22/2024‬‬ ‫اﻷﺳ ﺒﻮع اﻟﺨﺎﻣﺲ ‪geneticsdepartment@agr.asu.edu.eg‬‬ ‫‪3‬‬

 Let’s agree some SUBSTANTIAL RULES

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 Gene Cloning‫ﺧﻄﻮات‬
‫ ؟؟؟؟؟؟‬2.

DNA fragment

Vector

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 ‫ﺧﻄﻮات‪Gene Cloning‬‬
‫‪ 2.‬؟؟؟؟؟؟‬
‫‪Forward primer :‬‬
‫`‪5` ATGTCACAGGTTTGGCATA 3‬‬ ‫راﺟﻊ ﺗﺼﻤﻴﻢ‬
‫اﻟﺒﺎدﺋﺎت‬
‫‪Reverse primer :‬‬
‫`‪5` TTATTGAATCTTAGCCCCCT 3‬‬

‫ﻫﻞ ﻳﻤﻜﻦ اﺳﺘﺨﺪام ﻫﺬا اﻟﺰوج ﻣﻦ اﻟﺒﺎدﺋﺎت ﻓﻰ ﺗﺠﺮﺑﺔ الـ ‪ Cloning‬؟؟؟‬

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 ‫ﺧﻄﻮات‪Gene Cloning‬‬
‫‪ 1.‬ﺗﺤﻀﻴﺮ الـ ‪Insert of DNA‬‬
‫دـ( ﺗﺼﻤﻴﻢ اﻟ ﺒﺎدﺋﺎت ﻟﻠﺠﻴﻦ ﻣﺤﻞ‬
‫اﻟﺪراﺳﺔ‬
‫اﻟﺘﺘﺎﺑﻌﺎت اﻟﺨﺎﺻﺔ ﺑﺈﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ اﻟﻰ اﻟﺠﻴﻦ ﻣﺤﻞ‬ ‫‪ ‬إﺿﺎﻓﺔ‬
‫اﻟﺪراﺳﺔ ‪ :‬إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ‬
‫‪١‬ـ إﺿﺎﻓﺔ ﺗﺘﺎﺑﻊ إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ اﻟﻰ زوج اﻟﺒﺎدﺋﺎت‬

‫‪Type II‬‬
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 ‫إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻣﻦ اﻟﻄﺮاز‬
‫اﻟﺜﺎﻧﻲ‬
‫‪Restriction‬‬
‫اﻟﺜﺎﻧﻲ و ﻣﺼﺪرﻫﺎ ‪ ،‬و ﺗﺘﺎﺑﻊ‬ ‫ﺑﻌﺾ اﻷ ﻣﺜﻠﺔ ﻹ ﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻣﻦ اﻟﻄﺮاز‬
‫اﻟﺘﻌﺮف و ﻧﻬﺎﻳﺔ اﻟﻘﻄﻊ‬
‫‪Endonucleases‬‬
‫‪Type II‬‬

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 ‫‪ ‬إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻣﻦ اﻟﻄﺮاز اﻟﺜﺎﻧﻲ‬
‫‪ 1.‬ﻣﻮﻗﻊ اﻟﺘﻌﺮف ‪Recognition sequence‬‬
‫‪Palindromic‬‬

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 ‫ إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻣﻦ اﻟﻄﺮاز اﻟﺜﺎﻧﻲ‬
Recognition ‫ ﻃﻮل ﻣﻮﻗﻊ اﻟﺘﻌﺮف‬2.
4-cutters: sequence length
AluI 5´ ... AGCT ... 3´
MspI 5´ ... CCGG ... 3´

6-cutters
PvuII 5´ ... CAGCTG ... 3´
KpnI 5´ ... GGTACC ... 3´

8-cutters
NotI 5´ ... GCGGCCGC ... 3´

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 ‫ إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻣﻦ اﻟﻄﺮاز اﻟﺜﺎﻧﻲ‬
4-cutters:
Restriction site ‫ ﻣﻮﻗﻊ اﻟﻘﻄﻊ‬3.
^
AluI 5´ ... AG CT ... 3´
^
MspI5´ ... C CGG ... 3´

6-cutters
PvuII 5´ ... CAG ^CTG ... 3´
KpnI 5´ ... GGTAC^C ... 3´

8-cutters
^
NotI 5´ ... GC GGCCGC ... 3´
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 ‫‪ ‬إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻣﻦ اﻟﻄﺮاز اﻟﺜﺎﻧﻲ‬
‫‪ 4.‬ﻧﻬﺎﻳﺔ اﻟﻘﻄﻊ ‪Cleavage End‬‬

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 ‫ إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻣﻦ اﻟﻄﺮاز اﻟﺜﺎﻧﻲ‬
Cleavage End ‫ ﻧﻬﺎﻳﺔ اﻟﻘﻄﻊ‬4.
Sma I Blunt

Hind III Overhang′5

Kpn I Overhang′ 3

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 ‫ إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻣﻦ اﻟﻄﺮاز اﻟﺜﺎﻧﻲ‬
Cleavage End ‫ ﻧﻬﺎﻳﺔ اﻟﻘﻄﻊ‬4.
Sticky Ends
5' overhang 3' overhang

 phosphate group (in red ) are  phosphate group (in red ) are
exposed′ The 5 recessed′ The 5

 hydroxyl group (in blue ) are recessed′  hydroxyl group (in blue ) are exposed′
The 3 The 3
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 ‫• ﻣﺸﺎﺑﻬﺎت إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻣﻦ اﻟﻄﺮاز اﻟﺜﺎﻧﻲ‬

‫‪Isoschizomer‬‬ ‫‪Neoschizomer‬‬
‫‪s‬‬ ‫‪s‬‬
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 ‫‪ ‬ﻣﺸﺎﺑﻬﺎت إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻣﻦ اﻟﻄﺮاز‬
‫‪ 1.‬اﻟﺜﺎﻧﻲ‬
‫‪Prototype‬‬
‫أول إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ اﻟﺘﻰ ﺗﻢ اﻛﺘﺸﺎﻓﻬﺎ‬
‫‪ 2.‬ﻟﺘﺘﺎﺑﻊ ﻣﻌﻴﻦ‬
‫‪Isoschizomers‬‬
‫إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻟﻬﺎ ﻧﻔﺲ ﺗﺘﺎﺑﻊ ﻟﺘﻌﺮف و‬
‫‪3.‬ﻣﻮﻗﻊ اﻟﻘﻄﻊ‬
‫‪Neoschizomers‬‬
‫إﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻟﻬﺎ ﻧﻔﺲ ﺗﺘﺎﺑﻊ ﻟﺘﻌﺮف و ﻟﻜﻦ ﺗﺨﺘﻠﻒ ﻓﻰ‬
‫ﻣﻮﻗﻊ اﻟﻘﻄﻊ‬

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 ‫ﺧﻄﻮات‪Gene Cloning‬‬
‫‪ 1.‬ﺗﺤﻀﻴﺮ الـ ‪Insert of DNA‬‬
‫دـ( ﺗﺼﻤﻴﻢ اﻟ ﺒﺎدﺋﺎت ﻟﻠﺠﻴﻦ ﻣﺤﻞ‬
‫اﻟﺪراﺳﺔ‬
‫اﻟﺘﺘﺎﺑﻌﺎت اﻟﺨﺎﺻﺔ ﺑﺈﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ اﻟﻰ اﻟﺒﺎدﺋﺎت اﻟﻤﺴﺘﺨﺪﻣﺔ‬ ‫)‪1‬إﺿﺎﻓﺔ‬
‫ﻓﻲ الـ ‪PCR‬‬

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 Gene Cloning‫ﺧﻄﻮات‬
Insert of DNA ‫ ﺗﺤﻀﻴﺮ الـ‬1.
‫دـ( ﺗﺼﻤﻴﻢ اﻟ ﺒﺎدﺋﺎت ﻟﻠﺠﻴﻦ ﻣﺤﻞ‬
‫اﻟﺪراﺳﺔ‬
‫اﻟﺘﺘﺎﺑﻌﺎت اﻟﺨﺎﺻﺔ ﺑﺈﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻋﻦ ﻃﺮﻳﻖ اﺳﺘﺨﺪام الـ‬ ‫إﺿﺎﻓﺔ‬2)
 :Linker Linker
A synthetic, double-stranded oligonucleotide used to attach
sticky ends to a blunt-ended molecule.

Having blunt ends on both sides

Blunt ended DNA molecule

Contain the restriction site

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 ‫ﺧﻄﻮات‪Gene Cloning‬‬
‫‪ 1.‬ﺗﺤﻀﻴﺮ الـ ‪Insert of DNA‬‬
‫دـ( ﺗﺼﻤﻴﻢ اﻟ ﺒﺎدﺋﺎت ﻟﻠﺠﻴﻦ ﻣﺤﻞ‬
‫اﻟﺪراﺳﺔ‬
‫اﻟﺘﺘﺎﺑﻌﺎت اﻟﺨﺎﺻﺔ ﺑﺈﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻋﻦ ﻃﺮﻳﻖ اﺳﺘﺨﺪام الـ‬ ‫)‪2‬إﺿﺎﻓﺔ‬
‫‪Linker‬‬

‫‪BamH1‬‬

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 ‫ ﻋ ﻴ ﻮ ب إ ﺳ ﺘ ﺨ ﺪ ا م ا لـ‬
Linkers
Linkers DNA Fragment Linkers

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 Gene Cloning‫ﺧﻄﻮات‬
Insert of DNA ‫ ﺗﺤﻀﻴﺮ الـ‬1.
‫دـ( ﺗﺼﻤﻴﻢ اﻟ ﺒﺎدﺋﺎت ﻟﻠﺠﻴﻦ ﻣﺤﻞ‬
‫اﻟﺪراﺳﺔ‬
‫اﻟﺘﺘﺎﺑﻌﺎت اﻟﺨﺎﺻﺔ ﺑﺈﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻋﻦ ﻃﺮﻳﻖ اﺳﺘﺨﺪام الـ‬ ‫إﺿﺎﻓﺔ‬2)
Adaptor
 Adaptors :
A synthetic, double-stranded oligonucleotide used to
attach sticky ends to a blunt-ended molecule

sticky end
on one side
blunt end
on the other side

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 ‫ﺧﻄﻮات‪Gene Cloning‬‬
‫‪ 1.‬ﺗﺤﻀﻴﺮ الـ ‪Insert of DNA‬‬
‫دـ( ﺗﺼﻤﻴﻢ اﻟ ﺒﺎدﺋﺎت ﻟﻠﺠﻴﻦ ﻣﺤﻞ‬
‫اﻟﺪراﺳﺔ‬
‫اﻟﺘﺘﺎﺑﻌﺎت اﻟﺨﺎﺻﺔ ﺑﺈﻧﺰﻳﻤﺎت اﻟﻘﻄﻊ ﻋﻦ ﻃﺮﻳﻖ اﺳﺘﺨﺪام الـ‬ ‫)‪2‬إﺿﺎﻓﺔ‬
‫‪Adaptor‬‬

‫‪DNA Ligase‬‬

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 ‫‪ ‬ﻋ ﻴ ﻮ ب ا ﺳ ﺘ ﺨ ﺪ ا م ا لـ‬
‫‪Adaptors‬ﺔ ا ﻟ ﺘ ﺤ ﺎ م ﺟ ﺰ ﻳ ﺌ ﺎ ت‬
‫اﻣﻜﺎﻧﻴ‬
‫‪Adaptors‬ﻣﻌﺎ‬

‫ﺟﺰئ ال ‪ DNA‬ﻣﺎزال ذو ﻧﻬﺎﻳﺎت‬

‫‪3/22/2024‬‬
‫ﻣﺘﺴﺎوﻳﺔ‬
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 ‫‪ ‬ا ﻟ ﺘ ﺮ ﻛ ﻴ ﺐ ا ﻟ ﺼ ﺤ ﻴ ﺢ ﻟ ﻞـ‬
‫‪Adaptors‬‬

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 ‫ﺗﺤﻀﻴﺮ الـ ‪Insert of‬‬ ‫ﻫﻨﺪﺳﺔ‬
‫‪ DNA‬اﻟﺠﺰء اﻟﺜﺎﻧﻲ‬
‫وراﺛﻴﺔ‬

‫ﻗﺴﻢ اﻟﻮراﺛﺔ – ﻛﻠﻴﺔ اﻟﺰراﻋﺔ – ﺟﺎﻣﻌﺔ‬ ‫‪Email‬‬

‫ﻋﻴﻦ ﺷﻤﺲ‬
‫‪3/22/2024‬‬ ‫اﻷﺳ ﺒﻮع اﻟﺨﺎﻣﺲ ‪geneticsdepartment@agr.asu.edu.eg‬‬ ‫‪25‬‬
 ‫ﺗﺤﻀﻴﺮ الـ ‪Insert of‬‬ ‫ﻫﻨﺪﺳﺔ‬
‫‪ DNA‬اﻟﺠﺰء اﻟﺜﺎﻧﻲ‬
‫وراﺛﻴﺔ‬
‫‪Do you‬‬
‫‪have any‬‬
‫? ‪Question‬‬
‫ﻗﺴﻢ اﻟﻮراﺛﺔ – ﻛﻠﻴﺔ اﻟﺰراﻋﺔ – ﺟﺎﻣﻌﺔ‬
‫ﻋﻴﻦ ﺷﻤﺲ‬
‫‪3/22/2024‬‬ ‫اﻷﺳ ﺒﻮع اﻟﺨﺎﻣﺲ‬ ‫‪26‬‬
 Insert of ‫ﺗﺤﻀﻴﺮ الـ‬ ‫ﻫﻨﺪﺳﺔ وراﺛﻴﺔ‬
‫ اﻟﺠﺰء اﻟﺜﺎﻧﻲ‬DNA
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3/22/2024 geneticsdepartment@agr.asu.edu.eg ‫اﻷﺳ ﺒﻮع اﻟﺨﺎﻣﺲ‬ 27
 ‫ﺗﺤﻀﻴﺮ الـ ‪Insert of‬‬ ‫ﻫﻨﺪﺳﺔ‬
‫‪ DNA‬اﻟﺠﺰء اﻟﺜﺎﻧﻲ‬
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‫ﻋﻴﻦ ﺷﻤﺲ‬
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