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Understanding Molecular Cloning: Techniques and Applications

Molecular cloning replicates identical DNA molecules, commonly used to increase recombinant DNA amounts. DNA of interest is inserted into vectors and introduced into host organisms for replication. Explore various techniques, such as TA cloning, and applications like gene therapy and protein production.

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Understanding Molecular Cloning: Techniques and Applications

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  1. Molecular Cloning Kimberly Wright 21SEP2018

  2. Cloning? • Molecular cloning is the process of replicating identical DNA molecules • It is often used for increasing the amount of recombinant DNA • DNA sequences of interest (genes) are inserted into a vector/plasmid and introduced into a host organism where it can be replicated

  3. Bacterial Conjugation • Joshua Lederberg and Edward Tatum (1946)-demonstrated bacterial recombination in E. coli • Two different stains of E. coli that could not grow on media unless supplemented • Mixed the two stains and produced colonies https://www.ncbi.nlm.nih.gov/books/NBK21942/figure/A1306/?report=objectonly

  4. Fertility factor Cross 1: A- B- C+ D+StrR x A+B+ C- D-StrS Cross 2 : A-B-C+ D+StrS x A+B+ C- D-StrR Result: A+B+ C+ D+StrR • William Hayes (1952)- Demonstrated unidirectional gene transfer • Did crosses with streptomycin resistance and streptomycin sensitive • Streptomycin resistance transferred to only one cross • One was a donor and the other a recipient

  5. Stanley Cohen and Herbert Boyer • Showed that individual genes can be cloned and isolated by enzymatically fragmenting DNA molecules with ECor1 • They linked the fragments into bacterial plasmids and introduced the resulting recombinant DNA molecules into bacteria • This paved the way for molecular cloning

  6. What is a plasmid? • A small DNA molecule that is replicated separately from the genome and replicates independently • Typically bacteria use these molecules for antibiotic resistance or other beneficial survival reasons • Contains at least a gene of interest, multiple cloning site, and a selection gene First widely used cloning vector (Stanley Cohen and Herbert Boyer, 1977)

  7. Types of Plasmids • Fertility or F plasmids – Conjugational plasmids (E. Coli) • Resistance or R plasmids – antibiotic resistant • Col or colicin plasmids – proteins that kill other bacteria • Degradative plasmids - allow the host bacterium to metabolize unusual molecules • Virulence plasmids - confer pathogenicity on the host bacterium

  8. Cloning https://www.neb.com/tools-and-resources/feature-articles/foundations-of-molecular-cloning-past-present-and-future

  9. Blunt vs. sticky ends and ligases • Ligation of blunt ends are less efficient because it has to rely on association • Complementary sticky ends stick together and ligases seals the gap • Using ATP the 5’ end phosphate group is attached to the 3’ end hydroxyl group • Can put blunt ends on sticky ends • Requires turning the blunt end into a sticky end https://eclass.upatras.gr/modules/document/file.php/BIO276/Gene%20Cloning%20%26%20DNA%20Analysis.pdf

  10. TA cloning • Amplify DNA through PCR with Taq polymerase • Adds a A overhang to the 3’ end of the PCR product • The linearized vector has a 3’ T overhang that lacks a 3’ hydroxyl group to prevent the phosphodiester bond from forming https://en.wikipedia.org/wiki/TA_cloning

  11. Applications • Unlimited amount of DNA of interest • Gene Therapy • Understanding gene expression • Recombinant proteins • Produce antigens for testing vaccines • Produce insulin for diabetes • Create transgenic organisms

  12. References • Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000. Bacterial conjugation. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21942/ • Jayaraman, R. (2011). William Hayes and his Pallanza bomb shell. Resonance, 16(10), 911–921. https://doi.org/10.1007/s12045-011-0089-x • Biolabs, New England. “Foundations of Molecular Cloning - Past, Present and Future.” New England Biolabs: Reagents for the Life Sciences Industry, www.neb.com/tools-and-resources/feature-articles/foundations-of-molecular-cloning-past-present-and-future. • Brown, T.A. Gene Cloning and DNA Analysis: An Introduction. 6th edition. 2010 ISBN 978-1-4051-8173-0 Available from: https://eclass.upatras.gr/modules/document/file.php/BIO276/Gene%20Cloning%20%26%20DNA%20Analysis.pdf • Cohen, S. N., Chang, A. C. Y., Boyer, H. W., & Helling, R. B. (1973). Construction of Biologically Functional Bacterial Plasmids In Vitro. Proceedings of the National Academy of Sciences, 70(11), 3240–3244. https://doi.org/10.1073/pnas.70.11.3240 • Cohen, S. N. (2013). DNA cloning: A personal view after 40 years. Proceedings of the National Academy of Sciences, 110(39), 15521–15529. https://doi.org/10.1073/pnas.1313397110 • Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Isolating, Cloning, and Sequencing DNA. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26837/ • Holton, T. A., & Graham, M. W. (1991). A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Research, 19(5), 1156. https://doi.org/10.1093/nar/19.5.1156

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