The most widely used techniques

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Abstract

Gene cloning and recombinant DNA technology are the most widely used techniques that are used in the diagnosis of many diseases, production of proteins on a commercial scale, gene transfer as well as DNA amplification. The technique involves restriction, ligation and transformation as the principle steps. The plasmid vector (pUC19) and lambda (?) DNA were digested using restriction endonuclease, EcoRI. This restriction endonuclease cuts the ? DNA into six pices or fragments and linearizes the circular pUC19, producing sticky ends. Five fragments of ? DNA one fragment of linear plasmid were observed in agaros gel electrophoresis. The size of pUC19 fragment was found using the ? DNA fragment as the marker.

Table 3 shows the different number of colonies that were counted on different plates. Plate 1 contains two white colonies and no blue colonies,it is a control plate. This must be due to contamination of the plate. There were 212 blue colonies on plate 2 and no white colonies, this is unexpected as there must be some white colonies (Lodge,J. & Lund,P.,2007). The percentage of transformants there was zero and the transformant efficiency was 4452 transformants µg-1 DNA for this plate. Plate 3 contains 16 and 7 blue colonies respectively. The results were unexpected as there shouldnt be any blue colonies. This was because there was no ligase in that plate and there shouldnt be any ligation, so the blue colonies formed muse be due to contamination. Plate 4 showed 22 blue and 6 white colonies. The percentage of transformants were 22.42 %.

Introduction

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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. The phenomenon of restriction was discovered by Bertani and Weigle, who were growing bacteriophage lambda on different strains of E. coli. The molecular basis or restriction was discovered by Arber and Dussoix. 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. 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 (Lodge, J. & Lund, P., 2007). 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 (Howe, C. 1995). 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(Old & Primrose., 1989). There are now more than 1200 known restriction enzyme types. Different restriction enzymes cleave 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 (Lodge, J. & Lund, P., 2007). Sma1 always looks for the sequence CCCGGG, whereas HindIII cuts the sequence AAAGCTT, on the DNA double strands when read from 5'à 3' (Sofer, W. H. 1991).

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.

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).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. 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 (Lodge, J. & Lund, P., 2007).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 (Sofer, W. H. 1991). 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.

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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 the mixture of E. coli and the recombinant plasmid will ne cultured on specific antibiotic resistance (mostly ampicillin) 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).

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) 2009- 2010 from page 6 to12 with few things altered.

In restriction, sample loading was done in 5 wells while accordint to CMB practical booklet sample loading was done in 3 wells, with ligase + stopping mix and without ligase + stopping mix in the additional 2 wells.

RESULTS

Restriction digestion

The ? DNA and the pUC19 were restricted using EcoRI and the product was then observed on Agrose gel electrophoresis as a number of bands.

Fig-1. DNA and puc19 were digested using ecor1 and visualised through gel electrophoresis (0.8%). The gel was stained with ethidium bromide and observed on transilluminator. Well-1 contain dna was restricted by ecor1. Further well-2 contained undigested puc19. Whereas digested puc19 was present in well-3. In well-4 dna puc19 joined with t4 dna ligase was present. While wel-5 contained puc19 and dna without any ligase.

Discussion

Restriction digestion

Restriction of ? DNA was done using the restrintion endonuclease, EcoRI. The result obtained was analysed through agarose gell electrophoresis, producing the bands as in Fig 1. After restriction of ? DNA, 5 bands were produced. Different restriction endonucleases determines different number of fragments. ? DNA when cut with EcoRI gives 6 bands usually, but the bands observed were 5, so it might be possible that due to less difference in size of fragments i.e. 5.93 Kb and 5.54 Kb, therefore, it was assumed that two bands might have merged and appeared as one band. The fragments in well1 were not clear, this may be due to any practical error such as pippeting. Still 5 bands were visualised. The distance travelled by the fragments and their sizes correspond to the relationship between them. The larger the size of the fragment, less distance it will travel, as larger size occupies larger volume and so cannot travel efficiently within the pores of the gel (hartwell 2004). Due to some practical error, well 2 containing pUC19, which must be showing one or two bands, didn't showed any bands. Well 3 had a dark band showing the restriction of pUC19 with EcoRI. pUC19 travelled 29.0 mm, but as there were no bands in well 2 so it cannot be said that pUC19 was digested or not. There were two light bands observed in well 4, confirming the transformance of pUC19 containing fragment of ? DNA, that circularized using ligase and other fragment is the restricted ? DNA. They covered15 mm and 18 mm respectively. In the last well i.e. well 5, tow dark and three light bands were observed. One of them travelling the same distance as travelled by pUC19, so the remaining bands might be the ? DNA.

Table 3 shows the different number of colonies that were counted on different plates. Plate 1 contains two white colonies and no blue colonies,it is a control plate. This must be due to contamination of the plate. There were 212 blue colonies on plate 2 and no white colonies, this is unexpected as there must be some white colonies (Lodge,J. & Lund,P.,2007). The percentage of transformants there was zero and the transformant efficiency was 4452 transformants µg-1 DNA for this plate. Plate 3 contains 16 and 7 blue colonies respectively. The results were unexpected as there shouldnt be any blue colonies. This was because there was no ligase in that plate and there shouldnt be any ligation, so the blue colonies formed muse be due to contamination. Plate 4 showed 22 blue and 6 white colonies. The percentage of transformants were 22.42 %.

Refrences

  1. Brown, T.A. (2001). Gene cloning and DNA analysis.(4th ed.).Oxford:Blackwell Science
  2. Lodge, Julia, Lund, Pete & Minchin, Steve (2007). Gene cloning. New York: Taylor & Francis Group
  3. Sofer, W. H. (1991). Introduction to genetic engineering. USA: Reed publishing.
  4. Burrell, M. M. (1993). Enzymes of molecular biology. Newyork: Humna press.
  5. Turner, P., McLennan, A., Bates, A. & White, M. (2005). Instant notes: Molecular Biology. (3rd ed.). Abingdon: Taylor & Francis Group.
  6. Howe, C. (1995). Gene cloning and manipulation. USA: Press syndicate of university of Cambridge.
  7. Old, R. W. & Primrose, S. B. (1989). Principles of gene manipulation:An introduction to genetic engineering.( 4th ed.).London: Blackwell.
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