Method to extract and purify human genomic DNA

Abstract: Method to extract and purify human genomic DNA, which provides for the following steps: charging the sample, which undergoes a lysis treatment extracting with chloroform or other organic solvents centrifuging to eliminate the protein portion diluting in water and precipitating with cationic detergent filtering and adhering of the genomic DNA to a suitable filter washing the filter and eluting the human genomic DNA which is re-suspended in water.

Description: This invention concerns a method to extract and purify genomic DNA from human total blood, tissues, sperm, cultured cells. The same method can be used also for extracting DNA from animal blood and tissues, virus (es. hepatitis), vegetal cells. To be more exact, the invention provides for a method which can be partly reproduced manually and partly automatically in limited performance times. The traditional method employed, for instance, by the "DNA Extractor" apparatus of the Applied Biosystem is known but is not satisfactory owing to its low percentage yield and its slowness; moreover, it employs as a reagent phenol which is expansive, irritant and neurotoxic. The present applicant has therefore tackled the problem of providing a method which enables higher percentage yields to be achieved more speedly and simply. Known from Honig et al, Research in Molecular Biology, Volume 3, 1974, pages 93-166, are other effective methods for extracting and purifying genomic DNA, using, for example, the cationic detergent cetyltrimethylammonium bromide. The method to extract and purify human genomic DNA is set forth and characterized in the main claim, while the dependent claims describe variants of the idea of the main solution. The method according to the invention makes it possible to obtain in about twenty minutes what can be obtained otherwise in about four hours with a lower yield. Moreover the method according to the invention can be automated at least partly with a suitable apparatus which provides the human genomic DNA in 10 to 15 minutes in a test-tube containing a small quantity of water (500-1000 microlitres for instance). The method, according to the invention, for the extraction and purification of DNA, starting from human blood as a whole, provides for at least the following steps: charging the sample, which undergoes a lysis treatment extracting the protein with an organic solvent, chloroform for instance centrifuging to eliminate the protein portion diluting in water and precipiting with cationic detergent filtering and adherence of the genomic DNA to a suitable filter washing the filter and eluting the genomic DNA, which is resuspended in water. The DNA obtained in this way can be employed in futher analysis: 1. Enzimatic restriction-Southern blot. 2. Amplification of specific genic fragments with the polymerase chain reaction (PCR). Percentage yields as compared to the traditional methods vary between 120% and 150%, with a recovery of 40 micrograms per millilitre of blood. To be more exact, the method according to the invention includes the following main steps: STEP 1: charging a sample of blood, normally between 300 and 2500 microlitres but also a different amount, which undergoes a treatment of lysis by a cationic detergent for example tetradecyltrimethilammoniumbromide (TTAB) or dodecyltrimethilammoniumbromide (DTAB) both with sodium cloride at a concentration higher than 0.5 M. This solution is mixed and heated up to 68°C and incubated for a while (es. five minutes). STEP 2: an extraction is made by adding 1 volume of chloroform or other organic solvents. STEP 3: the mixture undergoes centrifuging with a normal bench centrifuge for some minutes to eliminate the protein portion which forms a clog with the organic phase. STEP 4: after centrifuging, to the acqueos phase are added a quantity of water to decrease the ionic strenght below 0,5 M NaCl, and a cationic detergent (for instance a solution of 5% of Cetyl-trimetil-ammonium bromide), so that precipitation of the cationic detergent micellar complex-DNA takes place after a short mixing operation. STEP 5: the solution thus obtained and containing the micellar-DNA complex then undergoes filtration. The ultrafiltration takes place with a filter (for instance, sintered borosilicate glass or an organic matrix like polypropilene or polyethylene) of known and tested porosity (pore size between 5 and 15 micron), which retains the DNA-cationic detergent micellar complex in a satisfactory way. The hydrophilic surface enables DNA to be recovered easily and speedly after the washing operations. The organic matrix filter allows a slower recovery of DNA so it is not commonly used at the moment. As a genomic DNA is immobilized on the filter, it is then eluted. STEP 6: this elution takes place after a series of washing of the filter: a first wash is carried out in an acqueos solution of a low ionic strenght to eliminate the excess of detergent; a second wash is carried out with a mixtures of alcohol and an ionic solution, for instance of the type NaCl 0,2 M/70% ethanol, to change the DNA-cationic detergent complex into DNA-Na; a third wash is carried out with a mixture of alcohol and a solution of low ionic strenght to remove the excess of salts, for instance NaCl type (for instance, 70% ethanol and 30% water). After having removed every trace of alcohol from the filter by a current of air, the DNA is recovered by washing the filter with an acqueos solution of a low ionic strenght (water, for instance) at room temperature or by heating, for instance at 68°C.

Claims: Method for extraction of genomic DNA from a material selected from whole blood, tissues, sperm, cultured cells, virus and vegetal cells, said method comprising the steps of: 1- subjecting a sample of said material to a lysis treatment by a first cationic detergent, selected from tetradecyltrimethylammoniumbromide and dodecyltrimethylamoniumbromide, with sodium chloride at a concentration higher than 0.5 M, followed by heating up to 68°C and incubation for about five minutes; 2- adding to the solution obtained in step 1 an organic solvent to extract the proteins contained in said sample; 3- centrifuging the mixture obtained in step 2 in a bench centrifuge to separate an organic phase containing proteins and an aqueous phase; 4- diluting said aqueous phase with water up to an ionic strength below 0.5 M of sodium chloride, and adding a second cationic detergent, thereby a cationic detergent micellar-DNA complex is formed; 5- separating said cationic detergent micellar-DNA complex by filtration with a filter with pore size from 5 to 15 micrometers; 6- washing said filter by carrying out a first wash with an aqueous solution of a low ionic strength to eliminate the excess of detergent; a second wash with a mixture of alcohol and a solution of sodium chloride, to change said cationic detergent micellar-DNA complex into DNA-Na; a third wash with a mixture of alcohol and a solution of low ionic strength to remove the excess of salts, and then removing of alcohol traces from said filter by a current of air and re-suspending of the DNA by washing the filter with an aqueous solution of low ionic strength at room temperature or by heating up to 68°C. Method according to claim 1, characterized in that said material is whole blood. Method according to claim 1, characterized in that said organic solvent added in step 2 is chloroform. Method according to claim 1, characterized in that said second cationic detergent added in step 4 is a solution of cetyl-trimethyl-ammonium bromide. Method according to claim 4, characterized in that said solution of cetyl-trimethyl-ammonium bromide has a concentration of 5%. Method according to claim 1, characterized in that said filter is made of sintered borosilicate glass. Method according to claim 1 characterized in that said filter is made of an organic matrix selected from polypropylene and polyethylene. Method according to claim 1, characterized in that said second wash in step 6 is carried out with a mixture of sodium chloride 0.2 M/70% ethanol. Method according to claim 1, characterized in that said third wash in step 6 is carried out with a mixture of 70% ethanol and 30% water. Method according to claim 1, characterized in that said re-suspension of the DNA of step 6 is carried out by washing said filter with water.

Inventors: SCHNEIDER CLAUDIO, SCHNEIDER, CLAUDIO
Applicants: TALENT SRL
Application year: 1990
Main classification: C07H1
IPC codes: C12N15