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Molecular Cloning

Molecular Cloning. Kimberly Wright 21SEP2018. Cloning?. Molecular cloning is the process of replicating identical DNA molecules It is often used for increasing the amount of recombinant DNA

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Molecular Cloning

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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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