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    Site Directed Mutagenesis

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    Sophie Tran
    A molecular biology technique in which a mutation is created at a defined site in a DNA molecule, usually a circular molecule known as a plasmid. Simply done by designing a 3mutated' primer, followed by PCR.

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    Attribution Non-Commercial (BY-NC)
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    Site Directed Mutagenesis

    Uploaded by

    Sophie Tran
    210 views21 pages
    A molecular biology technique in which a mutation is created at a defined site in a DNA molecule, usually a circular molecule known as a plasmid. Simply done by designing a 3mutated' primer, followed by PCR.

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    A molecular biology technique in which a mutation is created at a defined site in a DNA molecule, usually a circular molecule known as a plasmid. Simply done by designing a 3mutated' primer, followed by PCR.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    210 views21 pages

    Site Directed Mutagenesis

    Uploaded by

    Sophie Tran
    A molecular biology technique in which a mutation is created at a defined site in a DNA molecule, usually a circular molecule known as a plasmid. Simply done by designing a 3mutated' primer, followed by PCR.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 21

    Vietnam National University-HCMC International University

    Site-directed mutagenesis protocol


    Lecturer : Dr. Tran Ngoc Duc

    L/O/G/O

    Groups members:
    Tran Thi Anh Thuy Tran Thi Dieu Thanh Than Thi My Linh BTIU08114 BTIU08110 BTIU08127

    Introduction

    What is Site-directed mutagenesis?

    A molecular biology technique in which a mutation is created at a defined site in a DNA molecule, usually a circular molecule known as a plasmid. Simply done by designing a mutated primer, followed by PCR.

    General procedure

    General procedure
    Purify template plasmid DNA from a dam+ Escherichia coli strain (to ensure that all GATC sites are methylated for later digestion with DpnI). Design forward and reverse primers that will bind to the region of DNA you want to mutate but that contain the modifications you wish to make. Run a primer-extension reaction with a proof-reading, non-displacing polymerase such as Pfu DNA polymerase. This results in nicked circular strands of the plasmid. Cut up the template DNA with DpnI. Transform the circular nicked DNA into a highly competent strain such as XL1-Blue. These cells will repair the nicks and not restrict the unmodified product DNA. Select colonies with the correct DNA.

    Site-directed mutagenesis protocol

    Experimental protocol
    1. Pick target amino acid to be changed 2. Design a synthetic oligonucleotide to mutate the target amino acid 3. Use this primer to synthesize double stranded DNA 4. Verify production of the mutation 5. Characterize the new enzyme

    Stratagene protocol
    This is the protocol for site-directed mutagenesis based on the stratagene kit Materials: Pfu turbo 10X Pfu turbo buffer dNTPs (10mM) Forward and reverse primers (0.1ug/l, see methods section for design tips) dH2O Dpn1 Competent cells

    Methods
    For oligo-design or Primer Design: Both primers must contain the mutation. * The mutation should be in the middle of the primer. Primers should be 25-45 nucleotides long and have a GC content of at least 40%. The melting temperature (Tm) should be 78C. The 3-end of the primer has to end on an C or a G.
    5 3 * 3 5

    Reaction
    Set up as follows:
    Components Template DNA (50ng/ l) 10X Buffer Forward Primer (0.1 g/ l) Reverse Primer (0.1 g/ l) dNTPs (10mM) Pfu turbo dH2O Amount 1 l 5 l 1l 1 l 1 l 1 l 40 l

    PCR program
    Depending on the length of the plasmid, this program can become very long, so it may be best to turn overnight 1. 950C for 1 minute 2. 950C for 50 seconds, 600C for 50 seconds, 680C for 1 minute/kb of plasmid length repeat this step 17 times, or 18 cycles total 3. 680C for 7 minutes 4. 40C hold

    Following PCR
    DpnI Digest: Add 1 l of DpnI to the reaction. Incubate at 37 C for 1-2 hour to digest parental DNA Run 5L of the digested reaction on a gel and compare to the undigested parental plasmid there should be some difference in band pattern. Transformation: Transform the nal reaction into competent cells

    clip

    Discussion

    Trouble Shooting
    If no product is seen, try repeating the protocol with 5% DMSO in the reaction mix. DMSO disrupts base pairing, facilitating strand separation in GC rich regions of DNA and reducing the propensity of the DNA to form secondary structure. The end effect, is a little DMSO will often get you past issues with poor primer design and/or difcult templates.

    Verifying the mutation


    Gene sequencing
    sequence the DNA to verify that the base changes have been introduced

    Restriction mapping
    use the creation of a new restriction site or the elimination of an existing one to verify the mutation

    Restriction screening
    Allows selection of mutant enzymes through the creation or elimination of a restriction endonuclease site

    The creation or elimination of a site changes the size of the DNA fragments obtained

    References
    L Zheng, U Baumann, and JL Reymond. An efcient one-step site-directed and sitesaturation mutagenesis protocol. Nucl. Acids Res., 32:e115, 2004 QuikChange Site-Directed Mutagenesis Kit Retrieved May. 18, 20010 from http://www.jove.com/details.php?id=1135

    Thank You for your kind attention!


    L/O/G/O
    www.themegallery.com

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