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Cloning and rDNA (I)

Cloning and rDNA (I)

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Cloning and rDNA (I)

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  1. Cloning and rDNA (I) Dr. Abdulaziz Almalik National center for Biotechnology King Abdulaziz City for Science and Technology Office: 228-Building 17 (F) Tel: 4813154 aalmalik@kacst.edu.sa

  2. Objectives of this lecture By the end of this lecture you will be able to: • Understand the concept of recombinant DNA technology • Define terms related to cloning and DNA recombination • Realize the value of recombinant DNA technology

  3. Expression vector DNA of interest Palindrome Restriction Enzyme Recombinant DNA Delivery vector Expressio system Expressio system

  4. Recombinant DNA: is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together. Recombinant DNA technology: the tools that enable scientist to manipulate DNA to produce new genetic combinations that are of value to science, medicine, agriculture, and industry.

  5. 1 2 Isolation of gene of interest Integration into expression vector Transformation into host cells Growth of cells (fermentation) Isolation & purification of protein Formulation of protein product 3 5 6 4

  6. Isolation of Gene of Interest • The gene of interest is a small segment in the large DNA molecule • Restriction enzymes (restriction endonucleases): enzymes that cut DNA strands at a specific sequences called restriction sites. When they cut DNA, restriction enzymes can form: - Sticky ends: stretches of unpaired nucleotides in the end of a DNA molecule that can form hydrogen bonds with complementary sticky ends. - Blunt ends: both strands terminate in a base pair. • Different restriction enzymes have different sequence specificities (palindrome)

  7. Examples of Palindromes Noon Murder for a jar of red rum مودته تدوم لكل هول *** وهل كل مودته تدوم

  8. Enzymes with wobbling cuts givesticky ends • HindIII - leaves 5´ overhangs 5’ --AAGCTT-- 3’ 5’ --A AGCTT--3’ 3’ --TTCGAA-- 5’ 3’ –TTCGA A--5’ • KpnI leaves 3´ overhangs 5’--GGTACC-- 3’ 5’ –GGTAC C-- 3’ 3’--CCATGG-- 5’ 3’ –C CATGG-- 5’

  9. Enzymes that cut at same position on both strands give blunt ends • SmaI 5’ --CCCGGG-- 3’ 5’ --CCC GGG-- 3’ 3’ --GGGCCC-- 5’ 3’ --GGG CCC-- 5’

  10. DNA Ligase • DNA ligation is the act of joining together DNA strands with covalent bonds with the aim of making new viable DNA or plasmid • T4 DNA ligase has the unique ability to join sticky and blunt ended fragments

  11. DNA Ligase • Sticky ends that are complementary can be ligated together even if they are produced by different restriction enzymes. • Sticky ends that are not complementary cannot be ligated together.

  12. BamHI -G GATCC- -CCTAG G- • BglII -A GATCT- -TCTAG A- • Result -GGATCT- -CCTAGA- No longer palindromic, so not cut by BamHI or BglII

  13. DNA Ligase can also join blunt ends DNA fragments with blunt ends generated by different enzymes can be ligated together (with lower efficiency), but usually cannot be re-cut by either original restriction enzyme. • SmaI -CCC GGG- • DraI-AAA TTT- • Ligations that re-constitute a SmaI or DraI site (CCCGGG or AAATTT) can be re-cut by SmaI or DraI. • Mixed ligation products (CCCTTT + AAAGGG) cannot be re-cut by SmaI or DraI. -CCCGGG- -AAATTT- -CCCTTT- -AAAGGG-

  14. You are now able to: • Understand the concept of recombinant DNA technology • Define terms related to cloning and DNA recombination • Realize the value of recombinant DNA technology