Methods in Molecular Biology and Genetic Engineering

Chapter 3 Methods in Molecular Biology and Genetic Engineering

Methods in Molecular Biology and Genetic Engineering

3.1 Introduction restriction endonuclease – An enzyme that recognizes specific short sequences of DNA and cleaves the duplex (sometimes at the target site, sometimes elsewhere, depending on type).

Methods in Molecular Biology and Genetic Engineering

3.1 Introduction cloning vector – DNA (often derived from a plasmid or a bacteriophage genome) that can be used to propagate an incorporated DNA sequence in a host cell.– Vectors contain selectable markers and replication origins to allow identification and maintenance of the vector in the host.

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3.2 Nucleases

nucleases hydrolyze an ester bond within a phosphodiester bond. phosphatases hydrolyze the ester bond in a phosphomonoester bond.FIGURE 01: The target of a phosphatase (a) and a nuclease (b). An endonuclease (c) and an exonuclease (d)

Methods in Molecular Biology and Genetic Engineering

3.2 Nucleases

endonuclease – Nucleases that cleave phosphoester bonds within a nucleic acid chain.– They may be specific for RNA or for singlestranded or double-stranded DNA.

exonuclease – Nucleases that cleave phosphoester bonds one at a time from the end of a polynucleotide chain.– They may be specific for either the 5′ or 3′ end of DNA or RNA.

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3.2 Nucleases Restriction endonucleases can be used to cleave DNA into defined fragments.

FIGURE 02: Restriction endonuclease

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3.2 Nucleases A map can be generated by using the overlaps between the fragments generated by different restriction enzymes.

FIGURE 03: A restriction map is a linear sequence of sites separated by defined distances on DNA

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3.3 Cloning Cloning a fragment of DNA requires a specially engineered vector. recombinant DNA – A DNA molecule that has been created by joining together two or more molecules from different sources. ligating (or ligation) – The process of joining together two DNA fragments.

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3.3 Cloning

subclone – The process of breaking a cloned fragment into smaller fragments for further cloning. MCS (multiple cloning site) – A sequence of DNA containing a series of tandem restriction endonuclease sites used in cloning vectors for creating recombinant molecules.

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3.3 Cloning

FIGURE 04: Plasmid transformation

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3.3 Cloning transformation – The acquisition of new genetic material by incorporation of added exogenous, nonviral DNA. Blue/white selection allows the identification of bacteria that contain the vector plasmid and vector plasmids that contain an insert.

FIGURE 05: E. coli colonies on agar plates with ampicillin, IPTG, and the color indicator X-gal

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3.4 Cloning Vectors Can Be Specialized for Different PurposesFIGURE 06: Several types of cloning vectors are available

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3.4 Cloning Vectors Can Be Specialized for Different PurposesFIGURE 07: A vector can be used in both yeast and bacteria

Methods in Molecular Biology and Genetic Engineering

3.4 Cloning Vectors Can Be Specialized for Different Purposes Shuttle vectors can be propagated in more than one type of host cell. Expression vectors contain promoters tha

t allow transcription of any cloned gene.

Methods in Molecular Biology and Genetic Engineering

3.4 Cloning Vectors Can Be Specialized for Different Purposes Reporter genes can be used to measure promoter activity or tissue-specific expression.FIGURE 10: Fluorescent proteins are powerful research tools

Courtesy of Joachim Goedhart, Molecular Cytology, SILS, University of Amsterdam.

FIGURE 09: A mouse promoter controls tissue-specific expression of lacZPhoto courtesy of Robb Krumlauf, Stowers Institute for Medical Research

Methods in Molecular Biology and Genetic Engineering

3.4 Cloning Vectors Can Be Specialized for Different Purposes Numerous methods exist to introduce DNA into different target cells.

FIGURE 11: DNA can be introduced into cells in several ways