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DNA extraction from clotted blood - DNA extraction from clotted blood using glycogen (Feb/14/2005 )

Hello,

I am trying to extract DNA from clotted blood and I found some protocols online that suggest treating the blood with glycogen. The glycogen is suppose to improve the yield. I was wondering if anyone could tell me specifically what the glycogen does? How does it help improve yields?

Thanks for your help.

-biogeek-

I think you can use Proteinase K to digest the clots. I am studying for clinical licensure and this is what it says to do in my study guide if you get a blood sample with clots. Unfortunately that's all it says. Good luck!

-TechKnights-

check this posting, good luck.

http://www.protocol-online.org/archive/2/2803.html

-billnew-

on emore link:
http://www.clinchem.org/cgi/content/full/44/8/1748
Technical Briefs

Optimized Procedure for DNA Isolation from Fresh and Cryopreserved Clotted Human Blood Useful in Clinical Molecular Testing
Luis A. Salazar, Mario H. Hirata, Selma A. Cavalli, Marcos O. Machado, and Rosario D. C. Hirataa

a address correspondence to this author at: Dept. of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, University of So Paulo, Av. Prof. Lineu Prestes, 580, CEP 05508900, So Paulo, SP, Brazil

In the routine clinical laboratory, large amounts of uncoagulated blood are collected and the blood clot usually is discarded. In molecular biology, cells from EDTA-anticoagulated or acid-citrate-dextrose-anticoagulated peripheral blood are used as sources of DNA (1)(2)(3). After leukocyte isolation, most procedures utilize enzymatic cell digestion, followed by extraction with hazardous organic solvents (phenol-chloroform) and precipitation with ethanol (4)(5). To minimize the volume of blood collected for laboratory tests, several authors have developed methodologies to isolate DNA from blood clots (4)(5)(6)(7)(8). However, the techniques may be difficult or impractical and may require slicing of the clot with scalpels or other sharp instrument, exposing laboratory personnel directly to contaminated blood (4)(7). Other techniques are time-consuming, using many chaotropic reagents, enzymes, RNA-removal steps, or large volumes of samples and reagents not suitable in the clinical laboratory (4)(5)(6)(7)(8).

We have optimized a nonenzymatic, nontoxic procedure for efficient DNA extraction from fresh and cryopreserved clotted blood.

Blood samples were obtained from 24 unrelated individuals who had given informed consent. We compared 10 paired samples of EDTA-anticoagulated blood and fresh blood clot and studied 14 samples of cryopreserved clot that had been frozen for 2 years at -20 C. Blood clots were homogenized with 9 g/L NaCl, using a Potter-MARCONI MA 099 system, for 30 s. The homogenizing Teflon probe was cleaned three times with 700 mL/L ethanol and 9 g/L NaCl between samples to avoid cross-contamination. One milliliter of each homogenized sample was centrifuged at 1200g for 5 min, and the supernatant was removed. Blood cells were lysed with 1 mL of Tris buffer 1 (10 mmol/L Tris-HCl, pH 8.0, 10 mmol/L KCl, 10 mmol/L MgCl2, 2 mmol/L EDTA, pH 8.0, and 25 mL/L Triton X-100). After centrifugation, the pellet was washed twice with Tris buffer 1 and lysed with 220 L of Tris buffer 2 (10 mmol/L Tris-HCl, pH 8.0, 10 mmol/L KCl, 10 mmol/L MgCl2, 2 mmol/L EDTA, pH 8.0, 0.4 mol/L NaCl, and 10 g/L sodium dodecyl sulfate) and incubated for 15 min at 56 C. Cellular proteins were removed by precipitation, after addition of 100 L of 5 mol/L NaCl. DNA was isolated by ethanol precipitation and solubilized in Tris-EDTA buffer (10 mmol/L Tris-HCl, pH 8.0, and 1 mmol/L EDTA).

The DNA concentration was measured by spectrophotometry at 260 nm, and DNA purity was determined by the A260/A280 ratio (3).

-billnew-

QUOTE (billnew @ Mar 3 2005, 10:07 AM)
on emore link:
http://www.clinchem.org/cgi/content/full/44/8/1748
Technical Briefs

Optimized Procedure for DNA Isolation from Fresh and Cryopreserved Clotted Human Blood Useful in Clinical Molecular Testing
Luis A. Salazar, Mario H. Hirata, Selma A. Cavalli, Marcos O. Machado, and Rosario D. C. Hirataa


Hi,

I've used the above procedure for DNA extraction from human blood of varying quality
and age and it works a treat. Ethanol precipitation is much easier than phenol! biggrin.gif
Clots can also be broken up once in lysis solution by gentle agitation and pipetting with a filter tip.

HTH.

--
B3ka.

-b3ka-

HELLO EVERYONE

i have blood samples that have been frozen for one year now ...do u think there is any chance to extract DNA from themand how?

-lula-

Thank you everyone for the useful information. If anyone else has some ideas on how to break up clots for a high throughput lab I am very interested in hearing them.

Lula, in regards to extracting from samples that have been stored for a year could you tell us at what temperature they have been stored at and are you planning on extracting from them manually? What type of anticoagulant are they stored in?

-biogeek-

hi


they are stored at 20 c on EDTA,,,,i extract DNA manually by home made solutions ,,,but i didnt try from frozen blood samples before ,,,though i heard that phenol extrction culd be of benifit
thanks for responding

-lula-