Molecular cloning are the experimental methods used to assemble the recombinant DNA. This involves replication of the DNA in the living organism. This is a method to produce exact replicas. Cloning was performed in a cell or a DNA. The recombinant DNA is inserted into a cell which has the ability to accept the foreign DNA. If the cell is able to accept the recombinant DNA it is called competency. If the cell naturally does not have the ability to accept the foreign DNA then these cells are forced to be competent by using calcium chloride or by electroporation method. The process of inserting or projecting a recombinant DNA into a host cell is called cloning.
Molecular cloning produces scientists with an unlimited quantity of the DNA segments derived from a genome and these segments can be used in a range of biological sciences such as gene expression, gene therapy and so on. By this methods many chemical reagents such as acrylic acid, ethylene glycol, methanol, ethylene oxide and salicylic acid can be produced. Recombinant DNA molecules are molecules formed by laboratory methods of genetic recombination to bring together genetic material from various sources. In general it is the combination of at least two strands of DNA. These molecules are possible because in all organisms DNA share a same chemical structure having a variation in the nucleotide sequence within the structure.
Cloning of a recombinant DNA involves the process of making copies of recombinant DNA by inserting into a vector DNA. Normally used vector DNA is pUC19 which has a high copy number. Molecular cloning involves four steps namely restriction, ligation, transformation and screening. Enzymes used for restriction are BamH1, Pst1, ECORI, and Xho1 which has unique restriction site and produce different sized fragments. By the use of recombinant DNA in cloning a specific combination of genes can be put into a vector DNA and then can be proliferated and expressed in a recipient cell.
Recombinant DNA technology is used in biotechnology, medicine and research. Some of the practical applications are found in agriculture, industry, food production, human and veterinary medicine such as recombinant insulin is synthesized by inserting the human insulin gene into E.coli or yeast which then produces insulin used in the treatment of diabetes.
Blue white screening is a method to identify the recombinant DNA. Vectors containing a ?-galactosidase gene(lacZ) can have complementation(?-complementation) with E.coli strain to form a b-galatosidase enzyme.
Alpha-complementation is the process of determining the transformation efficiency of the competent cell. Competent cells are E.coli cells that have the ability to accept the DNA we need to insert to them. The key factor of alpha-complementation is lacz gene product is a tetramer and each monomer is made up of lacZ? and lacZ?.
?-galactosidase enzyme is used in genetics and molecular biology as a detection marker in gene expressions. This enzyme can be split into peptides lacZ? and lacZ?. These peptides are inactive by themselves but when both are present together they act as a functional enzyme. When the DNA fragments are inserted into the vector the production of the lacZ? is disrupted and therefore the cells show no activity of ?-galactosidase.
The vectors and the host cells does not have the b-galactidase enzyme activity. LacZ gene has a multiple cloning site within itself which can be cut by different restriction enzymes. Therefore when a gene fragment is inserted into the vector, the lacZ gene is disrupted and cannot form beta-gatatosidase enzyme.
This enzyme is used in the blue white screening of the colonies. The presence or the absence of the b-galactosidase can be detected by X-gal. when X-gal is cleaved by the enzyme b-galactosidase it produces a characteristic blue dye, which make it easy for the identification of the cloned products in a plasmid. When a foreign DNA is inserted at the lacz? gene, the production of beta galactisodase is inhibited thereby lead to the formation of the white colonies. White colonies are those which have carried the recombinant DNA to the cells and has no enzyme function. Blue colonies are DNA which does not have the insert gene.
In this experiment we use unknown DNA samples, pMA and pMB will be digested initially and the restriction map has to be created And to subclone the fragment from the pMB into pUC19. This restricted insert DNA pMB is ligated with restricted pUC19 vector with the same enzyme and is transformed into the competent cell (E.coli XL1 blue) and analysed by electrophoresis.
Strains Ampicillin Tetracycline Kanamycin
DH5? – – –
pUC19 Resistant – –
pMA Resistant Resistant –
pMB – Resistant Resistant
XL1-blue – Resistant –
The strains of DH5?, PUC19, pMA, pMB, and XL1-blue are tested for antibiotic resistance in Luria-Broth agar plates and the results noted. pMA showed antibiotic resistance to ampicillin and tetracycline while pMB showed resistance to tetracycline and kanamycin. E.coli stain XL1-blue is resistant to tetracycline as well.
Restriction enzymes BAMH1 ECORI,
Fragment length Band1 Cm 1.4 1.4 1.5-1.6 1.4-1.5 1.4 1.6-1.7 1.4
Bp 4000 4000 3200 3600 4000 2800 4000
Band2 Cm – – 2.8 3.3 – 2.4-2.5 –
Bp – – 800 400 – 1200 –
Restriction enzymes BAMH1 ECORI,
Fragment length Band1 Cm 1.3 1.5-1.6 1.6-1.7 1.3-1.4 1.6-1.7 1.7 1.4-1.5
Bp 5000 3100 2900 4600 2700 2500 3700
Band2 Cm – 1.9-2 2.3-2.4 3.3 1.8 2.4 2.5
Bp – 1900 1300 400 2300 1300 1100
Band3 Cm – – 2.8 – – 2.4-2.5 –
Bp – – 800 – – 1200 –
The plasmids pMA and pMB was digested (single and double digest)with ther estriction enzymes BamH1, ECORI, pst1 and xhol. The results of the experiments showed that the xho1 had no restriction site in pMA and pst1 had two restriction sites in pMB. The plasmid pMB was found to be similar to the pMA with the 1.2kb, 0.8kb and 0.4kb fragments
The double digest with the
• Restriction enzymes BamHI and ECORI in the plasmids pMA and pMB had the same weight of 0.4kb
• Restriction enzymes ECORI and pst1, in pMA has the same weight (0.8kb) with one of the fragment restricted ECORI and pst in pMB
• Restriction enzymes BamH1 and pst1, in pMA has the same weight(1.2kb) with one of the fragment restricted by ECORI and pst1 in pMB.
The size of the restriction fragments of the plasmids pMA and pMB when digested with the enzymes was roughly obtained with the help of 1kb marker and these values were not accurate. As the pst1 has one restriction site in the pMA and two restriction sites in the pMB, we can assume that pMA is part of pMB. If the longer fragment of the pst1 site in the pMB when re-circled will have the same restriction enzymes sites as the pMA(BamH1,ECORI,pst1). The xhol restriction site in the pMB is not in the longer fragment and so this xhol site will not digest the longer fragment which is consistent with pMA.
The strains of DH5?, PUC19, pMA, pMB, and XL1-blue are tested for antibiotic resistance in Luria-Broth agar plates and the results noted. The bacterial host, DH5? has no resistance to any of the three bacterial strains, pUC19 is resistant to ampicillin, pMA is resistant to ampicillin and tetracycline, pMB is resistant to tetracycline and Kanamycin, and XL1-Blue is resistant to tetracycline. Therefore in the presence of an antibiotic cells containing recombinant dna and different plasmids could be selected by the growing host cells. For example, kanamycin can be used to select cells containing only pMB from a mixture of cells containing pMB and pUC19. As the results noted, pMA to be both ampicillin and tetracycline resistant whereas pMB to be resistant to tetracycline and kanamycin.
This may be because the tetracycline resistant gene is in pMA which is a part of the pMB, and the kanamycin resistant gene is in the Pst1 fragment which is not in the pMA that is it is in the Xho1 fragment which is not present in the pMA. The ampicillin resistant gene in the pMA which might be located in the pst1 restriction site which is insertion inactive that is when insert the pst1 fragment to pMA to become pMB therefore, pMB does not have ampicillin resistant gene