Micro Isolation And Analysis Of Plasmid DNA Biology Essay

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Gene cloning using Recombinant DNA technology has revolutionized science making it possible to manipulate DNA in the laboratory and insert it into living organisms where it resumes its normal functions. Formation of recombinants occurs via a range of processes that include isolation and subsequent purification of plasmid DNA, restriction digestion, ligation and transformation. The present study focuses on the development of recombinants using λDNA and plasmid DNA (pUC19) in the selected vector (Escherichia coli). The enzyme employed for restriction digestion is EcoRI (restriction endonuclease) and for ligation is T4 DNA ligase (ligase). The success of the transformation and recombination technique is determined by the formation of blue colonies (transformed bacteria) and white colonies (recombinant bacteria). Agarose gel electrophoresis aids in the separation and analysis of DNA. Based on the distinct bands visualized in the presence of Ultraviolet radiation, the molecular weight of the isolated plasmid DNA (pUC19) can be determined. Growth of colonies on the petri plates indicated successful transformation along with the formation of recombinant bacteria. Five distinct bands of λDNA were visualized after the agarose gel electrophoresis, based on which the molecular weight of pUC19 was determined.

INTRODUCTION:

Gene cloning or DNA cloning may be defined as the "synthesis of multiple copies of a chosen DNA sequence using a bacterial cell or another organism as a host" (BioBasics, 2007). The desired gene is incorporated into a vector and the resulting recombinant DNA molecule is then multiplied in an appropriate host cell. To carry out gene cloning successfully various steps such as isolation of the DNA and its further purification, cutting of the DNA by using enzymes known as restriction endonucleases, ligation of the DNA fragments and finally introduction of the hybrid DNA into the host cell, are involved.

The vector employed in this experiment and commonly used in Escherichia coli, is pUC19. It is isolated from E.coli strain DH5-α by various standard procedures. The method used for isolation of the plasmid DNA in this practical is the commonly used technique developed by Birnboim and Doly which involves alkaline lysis.

The basis of alkaline lysis is that there exists a narrow pH range wherein the denaturation of only the non-supercoiled DNA and not the supercoiled DNA occurs. On the addition of sodium hydroxide to the extract, the pH is set to 12-12.5 causing the hydrogen bonds in the non-supercoiled DNA to break, the double helix to unwind and the separation of the two polynucleotide chains. When the acid is added the denatured DNA strands reaggregate which is then pelleted by centrifugation. The pure plasmid DNA is separated as the supernatant and is further precipitated by 70% ethanol.

The restriction step involved uses restriction endonucleases such as EcoRI, which cuts the plasmid DNA at the hexanucleotide 5'-GAATTC-3' producing sticky 5' overhangs. On digestion with this enzyme, a reproducible set of fragments with predictable sequences are obtained. Once treated with restriction endonuclease, the DNA fragments are examined by agarose electrophoresis for the determination of their sizes. The obtained bands of DNA are envisaged by soaking the gel in a solution of ethidium bromide which intercalates between DNA base pairs and fluoresces when activated with Ultraviolet radiation. The migration of the DNA is plotted against log of size which gives a straight line in the lower size region.

DNA ligases are employed to join the fragments generated by the treatment with restriction endonuclease. The enzyme which is widely used for this reaction is T4 DNA ligase and the energy required for the reaction is supplied by Mg+ and ATP. Recircularisation of the plasmid DNA may be avoided by treating the restricted plasmid with alkaline phosphatise which removes the terminal phosphate groups. As a result, the free ends are only ligated to DNA fragments which are to be inserted. The T4 DNA ligase acts by synthesising two phosphodiester bonds between the unlinked nucleotides, one in each of the two strands of the double stranded molecule.

Transformation is the uptake of 'naked' DNA from the media by bacterial cells. The frequency of uptake of the DNA by bacterial cells, especially E.coli is not very high and therefore the most common method used in this experiment is suspension of the cells in high concentration of Calcium chloride and heat shock. The recombinant plasmids can be distinguished from a non-recombinant plasmid by detecting insertional inactivation of the lacZ gene of the plasmid resulting in the loss of its β-galactosidase activity. A histochemical test is carried out with a compound known as X-gal which is converted into a blue product by the enzyme, β-galactosidase. On addition of X-gal along with ampicillin to the agar, the non-recombinant plasmids which have a functional β-galactosidase activity will be coloured blue, whereas the recombinant cells which have a disrupted lacZ gene and hence do not synthesise the enzyme will be white in color. This system is referred to as Lac selection.

The experiment aims at obtaining clones of E.coli carrying parts of λ DNA inserted into the plasmid pUC19 by using the basic procedures in recombinant DNA technology which are isolation, purification, restriction, ligation and transformation.

MATERIALS AND METHODS:

Followed as per the prescribed practical guide, "Level M, Practical Booklet, Core Molecular Biology, 2010-2011".

RESULTS:

The result of the agarose gel electrophoresis is depicted in the figure:1 below.

Lanes 1 2 3 4 5 6 7 (-ve terminal)

(+ve terminal)

Figure: 1 Agarose gel electrophoresis sample results. Lane:1 DH5α, Lane:2 DH5α +pUC19, Lane:3 EcoR1 + λ DNA, Lane:4 Unrestricted pUC19, Lane:5 EcoR1+pUC19, Lane:6 Restricted pUC19+Restricted λ DNA + Ligase and Lane:7 Restricted pUC19+Restricted λ DNA - Ligase.

A smear of DNA was observed in Lane 1. Lane 2 also showed the presence of a smear along with a very faint band of isolated pUC19. Five distinct bands of λ DNA were observed in Lane 3 in contrast to the expected 6 bands. Lane 4 exhibited quite a series of bands of unrestricted pUC19, whereas Lane 5 showed a thick band of restricted pUC19. No bands were observed in Lane 6 and Lane 7.

SR NO.

DISTANCE MIGRATED (mm)

MOL.WT. OF RESTRICTED λ IN kb

MOL.WT. OF RESTRICTED λ IN LOG kb

1

35

21.8

1.338

2

42

7.52

0.876

3

46

5.93

0.773

4

48

5.54

0.744

5

55

4.8

0.681

GRAPH 1: Distance migrated by each v/s log molecular weight of restricted λ DNA

Sl no. of the plates

Tube

Sample added

No. of white colonies

No. of blue colonies

1

1

Negative control (DH5α)

0

0

2

2

DH5α +PUC19 (undiluted)

5

55

3

2

DH5α+ PUC19 (diluted)

1

3

4

3

DH5α + Restricted λ+ Restricted pUC19- ligase

1

0

5

4

DH5α + Restricted λ+ Restricted pUC19+ligase (undiluted)

16

62

6

4

DH5α + Restricted λ+ Restricted pUC19+ ligase) (diluted)

7

9

TABLE 1: OBSERVATION OF BACTERIAL TRANSFORMATION EXP.

GRAPH 1: Distance migrated by each v/s log molecular weight of restricted λ DNA

DISCUSSION:

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