The practice of inserting new genes into a cell is known as DNA manipulation or Gene manipulation. The techniques involved in the manipulation of gene is known as Recombinant DNA technology. The ingredients of this technology involves three major steps restriction or digestion, ligation anf transformation. (Nathans, D. and Smith, H.O. (1975) Ann. Rev. Biochem. 44, 273-293.
- Luria, S.E. and Human, S.L. (1952) J. Bacterial. 64, 557-569.
- Bertani, G. and Weigle, J.J. (1953) J. Bacterial. 65, 113-121.)
The phenomenon of restriction was discovered by Bertani and Weigle, who were growing bacteriophage lambda on different strains of E. coli. Meselson M., Yuan R. (1968). DNA restriction enzyme from E. coli. Nature 217: 1110-4.
The molecular basis or restriction was discovered by Arber and Dussoix. ( Book SeriesMethods in Molecular Biology ISSN1064-3745 (Print) 1940-6029 (Online) VolumeVolume 16 BookEnzymes of Molecular Biology PublisherHumana Press DOI10.1385/0896032345 Copyright1993 ISBN978-0-89603-322-1 (Print) 978-1-59259-503-7 (Online) DOI10.1385/0-89603-234-5:107 Pages107-200 Subject CollectionBiomedical and Life Sciences SpringerLink DateSunday, December 14, 2008)
Restriction involves cutting into fragments the DNA from one cell and introducing the fragment of interest into a host cell, usually E. coli. The fragment OF INTEREST cannot be simply introduced into the host cell or organism. To ensure that the piece of DNA is copied and passes on, a vector is required. These are small circular DNA molecules found in many bacteria. They have an origin of replication directing the replication of plasmid. (cr restriction)The vector will ensure that it is copied eachtime the cell copies it's own DNA and also that the copy is passed on to the next generation or the daughter cells at cell division. Plasmids which are used for cloning called vectors such as pBR322, pUC19. (Gene Cloning Lodge, Julia Minchin, Steve Lund, P. )
During restriction the vector(plasmid) for example pUC19 and the host cell (e.g. E. coli) are cut with the same restriction enzyme. Restriction enzymes are the part of the defence system of the bacteria against the incoming DNA, which may be from virus or plasmid from a foreign population of cells. .(gene cloning and manipulation).
Restriction endonucleases recognize a specific double stranded DNA sequence and cleaves DNA in both strands. These sequences are the palindromic sequences, it means that the sequences are same from both sides if it is read from 5'à 3'. The endonuclease cut the DNA in two different ways such that producing sticky ends like EcoRI and blunt ends like SmaI(Singh, B.D. (2004). The text book of biotechnology . Varanasi: Kalyani). there are now more than 1200 known restriction enzyme types. Different restriction enzymes cleaves different sequences. EcoRI is systhesized by the bacterium E.coli and so named after it, EcoRI. It cuts the DNA whenever it finds the sequence GAATTC. (Gene Cloning Lodge, Julia Minchin, Steve Lund, P.) (Title: Gene Cloning Author(s): Lodge, Julia; Minchin, Steve; Lund, P. Print ISBN: 9780748765348 eISBN: 9780203967287 Publisher: Taylor & Francis Publication Date: 01 Sep, 2006 Dewey Code: 572.8633; 660.6/5 Download Size: 4,170kB Total Pages: 473)
Sma1 always looks for the sequence CCCGGG, whereas HindIII cuts the sequence AAAGCTT, on the DNA double strands when read from 5'à 3'. (understanding genetic engineering.)
Restriction is usually carried out at 37°C in the presence of small volume of buffer and salt. Restriction enzymes are active over a wide range of conditions. Suitable conditions for each restriction enzyme have been determined. Restriction enzyme works in the presence of buffer, salt and magnesium chloride. (CR_RESTRICTION)
After describing the way of restriction, the next step is to join them together in a new combination. The process is known as DNA ligation. Joining together of the linear fragments of DNA with covalent bonds is known as DNA ligation. The process creates phosphodiester bonds between the 3' hydroxyl of one nucleotide and the 5' phosphate of the other one. ( Brown, T.A. (2006). Gene cloning and DNA analysis. (5 th ed.). Oxford: Blackwell ). The reaction of bond formation is usually catalysed by the DNA ligase enzyme. It joins the DNA fragments with sticky ends as well as blunt ends. The reaction requires energy. The most common DNA ligase is produced by a T4 bacteriophage and so the enzyme is calles T4 DNA ligase. It uses ATP as the sourse of energy. T4 DNA ligase is incubated at 16 C. It also operates best at the same temperature however it is active ata broad range of temperatures.The two components of DNA must be equimolar. .( DNA_Ligation). Once the foreign piece of DNA is ligated into a circle along with the plasmid, the essence of cloning has been carried out, and is also the desired outcome. But this is not the only ligation possibility, there are at least two other possibilities: one is the circularization of the original plasmid DNA and the other is that vector molecules may attach to each other reforming the plasmid or forming a larger molecule that contains more than one plasmid. These are undesirable so these must be avoided. (Title: Gene Cloning Author(s): Lodge, Julia; Minchin, Steve; Lund, P. Print ISBN: 9780748765348 eISBN: 9780203967287 Publisher: Taylor & Francis Publication Date: 01 Sep, 2006 Dewey Code: 572.8633; 660.6/5 Download Size: 4,170kB Total Pages: 473). Low concentration of foreign DNA favours the circularization of plasmid DNA, so by increasing the concentration of foreign DNA, so that the interaction among the molecules will be more frequent and so more recombinant molecules will be produced and are known as recombinant plasmid or recombinant DNA or r-DNA (introduction to genetic engineering). Usually one would like to ligate the foreign DNA or the gene of interest into the plasmid so that it is ready for bacterial transformation.
The completion of ligation takes us to the final step of the DNA manipulation i.e. transformation. It is the introduction of the r-DNA into E. coli. Transformation of completed in two steps. One is to get the r-DNA into the bacterial cell. As it is an inefficient process, so it requires selection of those cells which has successfully taken up the plasmid containing the gene of interest. So themixture of E. coli and the recombinant plasmid will ne cultured on specific antibiotic resistance (mostly ampicilin) agar plate. The bacterium that has taken up the recombinant plasmid will survive and will appear as white colony, while those without the gene of interest gives blue colony. This is known as blue-white screening. ( Turner, P., McLennan, A., Bates, A., White, M. (2005). Instant notes: Molecular
Biology. (3 rd ed.). Abingdon: Taylor & Francis Group.)
The aim of the experiment thus includes the isolation of the DNA of interest, use of restriction endonuclease EcoRI to cut the bacteriophage lambda and pUC19, anaylysing them by agros gel electrophoresis. Then ligating the plasmid pUC19 containing lambda fragment (the gene of interest) using DNA ligase enzyme also monitoring on mini-gel. The last step of the experiment is to transform into E.coli the recombinant plasmid pUC19. Isolating recombinant E. coli containing the gene of interest i.e. ampicilin resistance gene, through blue- white screening.
Materials and Methods
All the steps in the DNA manipulation experiment were followed exactly as in practical booklet (core molecular biology).