non organic dna extraction

danielg at uncmed.med.unc.edu danielg at uncmed.med.unc.edu
Mon Feb 19 08:34:23 EST 1990


Hi friends.  I had many requests concerning my posting on non-organic
extraction of DNA, so will now post my main reference.  For those of you
who wrote me and have not received a personal response, I tried but the
postman won't deliver.  Please excuse the typo's.  Reference for the
following is Nucleic Acids Research Vol 16 No 3 1988, p. 1215.

        One of the obstacles encountered when extracting DNA from a large
        number of samples is the cumbersome method of deproteinizing cell
        digests with the hazardous organic solvents phenol and
        isochloroform.  Several other non-toxic extraction procedures have
        been published, but require either expensive dialysis (1) or the
        use of filters (2).  A rapid, safe and inexpensive method was
        developed to simplify the deproteinization procedure.  This method
        involves salting out of the cellular proteins by dehydration and
        precipitation with a saturated NaCl solution.

        Buffy coats of nucleated cells obtained from anticoagulated blood
        (ACD or EDTA) were resuspended in 15 mL pp centrifue tubes with 3
        mL of nuclei lysis buffer ( 10 mM Tris-HCl, 400 mM NaCl and 2 mM
        Na2EDTA, ph 8.2).  The cell lysates were digested overnight at 37
        degrees with 0.2 mL of 10% SDS and 0.5 mL of a protease K solution
        (1 mg protease-K in 1% SDS and 2 mM Na2EDTA).

        After digestion was complete, 1 mL of saturated NaCl
        (approximately 6 M) was added to each tube and shaken vigourously
        for 15 seconds ( my note: will foam), followed by centrifugation
        at 2500 rpm for 15 minutes.  The precipitated protein pellet was
        left at the bottom of the tube and the supernatant containing the
        DNA was transferred to another 15 mL pp tube.  Exactly 2 volumes
        of room temperature absolute ethanol was added and the tubes
        inverted several times until the DNA precipitated.  The
        precipitated DNA strands were removed with a plastic spatula or
        pipette and transferred to a 1.5 mL microcentrifuge tube
        containing 100-200 uL TE buffer (10 mM Tris-HCl, 0.2 mM Na2EDTA,
        ph 7.5).  The DNA was allowed to dissolve 2 hours at 37 degrees C
        before quantitating.

        The DNA obtained from this somple technique yielded quantities
        comparable to those obtained from phenol-chloroform extractions.
        The 260/280 ratios were consistently 1.8-2.0, demostrating good
        deproteinization.  Restrictions were performed using a number of
        different enzymes requiring both high, medium or low salt
        concentrations, all resulting in complete restriction.  This
        procedure has been used in our laboratory on several thousand
        blood samples for parentage, population and forensic studies.
        This technique is used with our nonisotopic hybridization
        procedures (3) rendering the entire process of RFLP analysis free
        of toxic materials

        Rererences:

        1. Longmire, J., Albright, K., Lewis, A., Meincke, L. and
        Hildebrand, C. (1987) Nucleic Acids Res. 15, 859.
        2. Leadon, S. and Cerutti, P. (1982) Anal. Biochem. 120, 282-8.
        3. Dykes, D., Fondell, J., Watkins, P. and Polesky, H. (1986)
        Electrophoresis 7, 278-282.

I have used this successfully on whole blood, as well as cell culture
(HL60 cells) and frozen tissues (lymph nodes and tonsil).  Works great.
One poster asked me if it will work on fatty tissues, all I can say is try
it, I'm not sure, but the chloroform may have helped in the old method
(that's what we call p/c extraction around here!!).

The article noted that low salt restrictions work ok, but I have a friend
who has had some difficulty.  He has worked around this by adding a 70%
ethanol wash after the ethanol precipitation step, and, if restriction of
the resuspended DNA does not work (rare), he reprecipitates the DNA, and
resuspends.  Has never had any problem afterwards.

Regards!!

Daniel G. Sinclair
Research Tech II

__________________________________________________________________________
 Disclaimer: My opinion means | 'If you only knew how much I was holding
             nothing, but His |  back, you would commend me'
             means everything.|      - Charles Spurgeon, 19th century
                              |        evangelist (on humor in preaching)



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