Recombinant DNA Technology PDF

Recombinant DNA technology We by Dr.Ezat Mersal Describe recombinant DNA technology methods  Explain advantages OF r-DNA technology  shorte ❑ The word recombinant comes from the word combination, in which means the joining of tw...

Recombinant DNA technology We by Dr.Ezat Mersal Describe recombinant DNA technology methods  Explain advantages OF r-DNA technology  shorte ❑ The word recombinant comes from the word combination, in which means the joining of two things. gg ❑. DNA recombinant technology is a kind of technology that rejoining two DNA of different traits into a single DNA. jaf❑ Recombinant DNA technology can be defined as the formation of new combinations of heritable materials by insertion of foreign nucleic acid molecules into any virus, bacterial plasmids or other vector system  treatment disorders 119 j 0 N.  1- Desired DNA (foreign) =  2- Restriction endonuclease enzyme (RE)  3- Cloning Vector e.g. plasmid > S10 - j -  4- DNA ligase enzyme > - PNAS 29 Eis  5- Bacteria cell or Yeast cell > - PSI © 2015 John Wiley & Sons, Inc. All rights reserved. ❖ DNA segment (gene) separated by restriction endonucleases Interest gene Foreign Passenger Target Insert SI ❖ Unknown gene G mRNA RT DNA © 2015 John Wiley & Sons, Inc. All rights reserved.   Enzymes derived from Bacteria & fungi  Recognize & Cut DNA at specific site Ex  EcoR1 recognize & cut at GAATTC Eco  Bacterial own DNA is methylated © 2015 John Wiley & Sons, Inc. All rights reserved. Endonuclease  -P · 00  Vehicle that carry & introduce foreign DNA - fragments (gene) into a host cell see i Plasmid Cosmid Bacteriophage viruses © 2015 John Wiley & Sons, Inc. All rights reserved.  share  Small (To carry large DNA)  Well characterized  Posses non-essential = region for insertion target DNA + % No , as >  Recognize the site of cutting By RE - © 2015 John Wiley & Sons, Inc. All rights reserved.  ( ) 911095) : dig1Y X P Autonomous replication -1149  Carry a selectable marker Ampicillin resistance gene 10 3 /.. g's Y  No sex pilus © 2015 John Wiley & Sons, Inc. All rights reserved.  from bactiva A plasmid is a small DNA molecule = within a cell that is physically separated from a chromosomal DNA and can replicate independently. They are most commonly found in bacteria as small, circular, double- stranded DNA molecules 10-15 Kbp ?.. 6 Relaxed plasmid (2 Kbp) Introduced by Transformation ) Permeability (Calcium Chloride  Plasmid -S 9 [ii Plasmid. (COS) Cosmids = Cos + Plasmid ❑(COS) cohesive end of the lambda phage DNA ❑ Artificially constructed cloning Vector Introduced by Transduction 0 ❑ Useful for large DNA fragments (up to 40 Kbp) - Autonomous © 2015 John Wiley & Sons, Inc. All rights reserved. replication  Lambda phage of E.coli + DNA  Introduced by Transduction © 2015 John Wiley & Sons, Inc. All rights reserved.  a Vector for mammalian cell ❑ Plasmid ❑ Bacteriophage ❑ Cosmids ❑Virus be act as vehicle(Gene delivery vehicle) a❑Adenovirus, Vaccinia virus & Retrovirus : 9 % ga © 2015 John Wiley & Sons, Inc. All rights reserved. - sd  Cloning vector suitable for Eukaryotic is: P > ❑ Plasmid X ❑ Bacteriophage Y ❑ Cosmids Y Wa ❑ Animal viruses © 2015 John Wiley & Sons, Inc. All rights reserved.  - Ljs  Ligation by ligase enzyme S © 2015 John Wiley & Sons, Inc. All rights reserved.  -99s phos i + six Y Free from any restriction enzyme - 1) > - victor - 2) Easy to separate from cloning factor > - y) : = 3) No Sex pilus © 2015 John Wiley & Sons, Inc. All rights reserved.  tepy ❑ There are four methods in order to make r DNA which are : 1 - Transformation (bacterial and non bacterial) Vis & - &igjeifs 8 19539221 &, 2 - Phage Transduction (host) 15 sis gi - > - - % 3- Conjugation 9.4 - wis 14. e > 4- Transfection (applied on eukaryotic cells) -. P  bactiva or non bactiva I Transformation  Involvesthe insertion of gene of interest into a vector, and followed by the insertion or transformation of vector into the host organism such as E.coli. II Non-bacterial transformation Basically it involves the same procedure as normal transformation, but does not use any bacterial since the DNA or gene of interest is directly injected into the nucleus of the cell being transformed  Three methods to make r-DNA ins bacteria Vivas III Phage introduction  The r-DNA is made up by using viral infection instead of bacteria in a process named as transduction.  Phage introduction WRITTEN AND COMPILED BY COURSE COMMITTEE 21 shor emC A genomic library is a collection of the total genomic DNA from a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. O = ❑ Inthe medical field, rDNA approach helps in order to produce huge amounts of insulin, blood clotting factors (VIII & IX) , growth hormone. 8 a ❑ Production of recombinant vaccines. ❑ Advance technology also contributes in the effort to prevent and cure of sickle cell anemia and cystic fibrosis ❑ Reduce genetic disease in agriculture field in order to produce better crops. ❑ Alsohighly contributes in the gene therapy treatment. - C  Basic genetics : a human approach / BSCS. Dubuque, IA, Kendall/Hunt Pub. Co., c1999. 147 p. QH431.B305 1999  Genes, ethnicity, and ageing. Edited by Lincoln H. Schmitt, Leonard Freedman, Rayma Pervan. Nedlands, Australia, Centre for Human Biology, University of Western Australia ; Singapore, River Edge, NJ, World Scientific, c1995. 100 p.QH455.G45 1995  Genetic polymorphisms and susceptibility to disease. Edited by M. S. Miller and M. T. Cronin. New York, Taylor & Francis, 2000. 266 p.  GENETIC ANALYSIS AN INTEGRATED APPROACH Mark F. Sanders , John L. Bowman Second edition 2015 ISBN 978 0-321-94890-8 (student edition) www.pearsonhighered.com