Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Fundamentals of Molecular Biotechnology
Recombinant DNA Technology
Conclusion

Recombinant DNA technology encompasses a broad array of experimental Procedures that enable the isolation (cloning) of DNA fragments containing specific genes. The success of cloning relies on The ability to reproducibly cleave a DNA molecule into fragments of a defined size. Type II Restriction Endonucleases are utilized for precise DNA Cleavage. These Enzymes recognize specific nucleotide sequences and symmetrically cleave the phosphodiester bonds in each strand.

A typical Gene cloning experiment involves the following steps: 1. Restriction endonuclease Digestion of DNA isolated from the Organism that harbors the gene of interest. 2. Treatment of the cloning vector (typically a plasmid), capable of replicating within a host Cell, with the same restriction enzymes used to digest the donor DNA. 3. Mixing these two DNA samples and joining the fragments using T4 DNA ligase. 4. Transformation of host Cells with the ligated molecules. Amplification of the recombinant DNA within the transformed cells.

Specialized techniques are employed to select for cells containing recombinant DNA. To minimize The formation of self-ligated circular plasmid molecules catalyzed by T4 DNA ligase, the restricted plasmid DNA is treated with alkaline phosphatase to remove the 5'-terminal phosphate groups. The Selection of transformed cells harboring hybrid Plasmids involves: 1) testing for resistance to specific Antibiotics or utilizing colorimetric assays; 2) immunological assays or the detection of a specific protein—the product of the cloned gene; 3) Hybridization with a probe complementary to a region of the target gene.

To successfully clone an entire gene, the donor DNA is subjected to only partial digestion. This yields fragments of varying lengths, which are subsequently used to construct a genomic library. For the cloning of large DNA fragments, vectors based on Bacteriophages λ and P1, as well as F plasmids, have been engineered.

To obtain DNA fragments encoding eukaryotic Proteins, a complementary DNA (cDNA) strand is synthesized using purified mRNA as a template in a reaction catalyzed by Reverse Transcriptase; this single-stranded cDNA is subsequently used as a template for the Synthesis of the second strand. Following enzymatic Processing, this double-stranded complementary DNA is inserted into a vector.

Regardless of the specific cloning strategy employed, once the cloned sequence has been identified, it must be verified to ensure it represents a native structural gene.

References

Berger S. L., A. R. Kimmel (ed.). 1987. Methods in Enzymology, vol. 152. Guide to Molecular Cloning Techniques. Academic Press, London, United Kingdom.

Garfin D. E. 1995. Electrophoretic methods, p. 53—109. In J. A. Glasel and M. P. Deutscher (ed.). Introduction to Biophysical Methods for PROTEIN AND NUCLEIC Acid Research. Academic Press, San Diego, Calif.

Grinstead J., P. M. Bennett (ed.). 1988. Methods in Microbiology, vol. 21, Plasmid Technology. Academic Press, London, United Kingdom. Ioannou A. P., С. T. Amemiya, J. Games, P. M. Kroisel, H. Shizuya, C. Chen, M. A. Batzer, P. J. de Jong. 1994. A new bacteriophage-derived vector for the propagation of large human DNA fragments. Nat. Genet. 6: 84—89.

Kim U.-J., B. W. Birren, T. Slepak, V. Mancino, C. Boysen, H.-L. Kang, M. I. Simon, H. Shizuya. 1996. Construction and characterization of a human bacterial artificial chromosome library. Genomics 34: 213—218.

Leonardo E. D., J. M. Sedivy. 1990. A new vector for cloning large eukaryotic DNA segments in Escherichia coli. Bio/Technology 8: 841- 844. Martin C., L. Bresnick, R.-R. Juo, J. C. Voyta,

I. Bronstein. 1991. Improved chemiluminescent DNA Sequencing, BioTechniques 11: 110—114. Old R. W., S. B. Primrose, 1985. Principles of Gene Manipulation, 3rd ed. Blackwell Scientific Publications, Oxford, United Kingdom. Sambrook J., E. F. Fritsch, T. Maniatis. 1989. Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

Slightom J. L., R. F. Drong, Р. Р. Chee. 1993. Construction of λ clone banks, p. 121—146. In B. R. Glick and J. E. Thompson (ed.), Methods in Plant Molecular Biology and Biotechnology. CRC Press, Boca Raton, Fla.

Southern E. 1975. Detection of specific sequences among DNA fragments separated by gel Electrophoresis. J. Mol. Biol. 98: 503—507.

Winnacker E.-L. 1987. From Genes to Clones: Introduction to Gene Technology. VCH, New York, N.Y.

Review Questions

1. What are type II restriction endonucleases, and why are they so crucial for recombinant DNA technology?

2. Digestion of the circular double-stranded plasmid pCELI with various restriction enzymes and their combinations yields the following fragments (sizes are given in Base Pairs): EcoRI — 6.0; BamHI — 6.0; HindIII — 6.0; HaeII — 3.0, 2.0, and 1.0; EcoRI and HaeII — 2.0 and 1.0; EcoRI and HindIII — 3.5 and 2.5; EcoRI and BamHI — 4.5 and 1.5; BamHI and HindIII — 5.0 and

1.0; BamHI and HaeII — 3.0, 1.5, and 0.5; HindIII and HaeII — 3.0, 1.5, 1.0, and 0.5. Using these data, construct a restriction map of pCELI.

3. Describe the application of plasmid pBR322 as a cloning vector. What are its key features?

4. Describe the Main Properties of the pUC cloning system.

5. A clone library is typically generated by ligating a plasmid vector, subjected to complete digestion with BamHI, with chromosomal DNA partially digested with the restriction enzyme Sau3Al.

a. Why are two different enzymes used in this experiment?

b. WHAT IS A partial digest, and how is it performed?

c. Why is it frequently used to create clone banks?

6. Why is restricted plasmid DNA often treated with alkaline phosphatase prior to ligation?

7. Describe methods for introducing recombinant plasmids into a Gram-negative bacterium, such as E. coli.