RECOMBINANT DNA - Warner Pacific 250/Lecture 17 Recombinant...آ  RECOMBINANT DNA . Introduction •Recombinant

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  • RECOMBINANT DNA

  • Introduction

    • Recombinant DNA

    – A gene which has been constructed in the lab

    using two pieces of DNA from different biological

    sources

    – The technology used to create and study the

    molecules described

  • Cloning

    • Insertion of a gene or other DNA sequence

    into a bacterial cell, which will then be used

    as a factory to produce millions of copies of

    the gene of interest

  • Cloning

    • Steps in cloning:

    – Gene of interest is purified from cells or tissues

    – Restriction enzymes cut DNA

    – DNA fragments are ligated to a vector, such as a plasmid

    – Vector is transferred to a host cell

    – Host cell multiplies naturally into millions of cells, each

    containing copies of the DNA

    – Cloned DNA is purified from the host cells

    OR…

    – Gene product (protein) can be purified from host cells

  • Examples of Restriction Enzymes

  • Vectors

    • Acceptors and carriers of DNA to be cloned

    • Characteristics of vectors

    – Contain several restriction sites

    – Must be introduced into a host cell for replication of

    vector

    – Should have a selectable marker gene

    – May contain nucleotide sequences that allow for

    sequencing the inserted DNA

    • Vectors are usually modified bacterial plasmids

  • Example of Plasmid

  • How do we know the cells took up the plasmid?

    How do we know the plasmid contained the gene

    of interest?

  • Example of Recombinant Plasmid

  • Replicating Inserted DNA

  • Clicker Question: How do you know

    whether a host has taken up a vector

    during a typical cloning experiment?

    • A) It produces a blue colony

    • B) It produces a white colony

    • C) It produces any colony

    • D) It fails to grow at all

  • Clicker Question: What do you use to cut

    a plasmid?

    • A) An exonuclease

    • B) A restriction endonuclease

    • C) Tiny scissors

  • In Vitro DNA Amplification:

    Polymerase Chain Reaction (PCR) • PCR accelerated DNA duplication

    compared with cloning

    • Reaction components:

    – Taq DNA polymerase

    – Mg2+ ions

    – Deoxyribonucleotide triphosphates (A,T,C,G)

    – Primers specific for the DNA to be amplified

    – Template DNA

    – Optional: Reporter dye

  • In Vitro DNA Amplification: PCR

  • PCR Analysis

  • Reverse-Transcriptase (RT)-PCR

    • Start with mRNA

    – Isolate from tissue, cells, blood, plants,

    bacteria, etc.

    • Use reverse transcriptase to create cDNA

    – RT: viral enzyme

    – cDNA: complementary DNA

    • Amplify cDNA using PCR

    • Use to determine gene expression

  • After PCR… (possibilities)

    • Run the product (amplicon) on a gel

    • Compare CT values between groups

    • Purify the product

    • Sequence the product

    • Insert the product into a vector

  • Clicker Question: What is special about

    Taq polymerase?

    • A) It is especially fast at polymerizing

    • B) It can proofread better than other DNA

    polymerases

    • C) It can bend the space-time continuum

    • D) It can withstand high temperatures

  • Clicker Question: How does Dr. DuPriest

    use PCR in her research?

    • A) To create new vectors

    • B) To determine size of fragments

    • C) To compare gene expression between

    groups

    • D) To compare samples between a

    suspect and a crime scene specimen

  • Restriction Mapping

  • Uses of Restriction Mapping

    • Mapping a segment of DNA

    • Chromosomal analysis

    • Genetic testing (markers)

    • Forensic analysis – comparing DNA of

    different individuals

  • Blotting

  • Types of Blotting

    • Southern blotting

    – Measures presence / amount of specific DNA

    in a sample

    • Northern blotting

    – …specific RNA in a sample

    • Western blotting

    – …specific protein in a sample

    • All use specific probes to detect specific

    molecules (DNA/RNA/antibody)

  • DNA Sequencing

  • DNA Sequencing

  • DNA Sequencing

  • Clicker Question: Which type of

    blotting has largely been

    replaced by PCR?

    • A) Southern

    • B) Northern

    • C) Western

    • D) Eastern

  • Clicker Question: DNA

    sequencing has largely

    replaced…

    • A) Western blotting

    • B) Cloning

    • C) Restriction mapping

    • D) PCR