A simple and efficient non-organic procedure for the isolation of genomic DNA from blood

Abstract

The isolation of genomic DNA from blood typically involves digestion of nuclei with a combination of Proteinase K and SDS followed by deproteinization with organic reagents such as phenol and chloroform. Additional purification steps such as extensive dialysis, precipitation with a saturated solution of NaCl and/or absolute ethanol are then required for enzymatic analysis of the extracted DNA (1). The isolation procedure described here is both simple and rapid, eliminating the necessity for hazardous organic reagents. The method involves the incubation of nuclei with only Proteinase K at 65'C. It has been shown that Proteinase K is more active on denatured protein and that after prolonged incubation at 65°C it autoinactivates (2). As a result, following a 2 hour incubation, the extracted DNA can be used directly for enzymatic analysis without any additional purification. Nuclei were prepared by mixing an aliquot of whole blood with a 4 fold excess of ice cold CLB (0.32 M Sucrose, 10 mM Tris-HCl pH 7.6, 5 mM MgCl2, 1% Triton X-100). The lysate is centrifuged at 900 X g for 5 minutes at 4°C. The pellet is washed with CLB and following centrifugation the nuclear pellet is rinsed with a small aliquot of cold PLB (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 10 mM EDTA). For 1 ml of blood, the nuclei are resuspended in 250 ul of PLB+ ProK (PLB with 1 mg/ml Proteinase K) and incubated for 2 hours at 65°C. Periodic mixing during the 2 hour incubation is required for uniform lysis of the nuclear pellet and optimum recovery of DNA. A single 1.5 ml tube can be utilized for the entire procedure. The yield is greater than 90% of theoretical with an average size greater than 300 kb. DNA concentrations are determined spectrofluorometrically with Hoechst dye (3), or by electrophoresis with a series of known DNA samples on a 0.6% agarose gel stained with ethidium bromide. Conventional UV spectroscopy cannot be used since the proteolytic digestion products are not removed from the DNA. DNA isolated from blood is readily digestible with all restriction enzymes tested (Fig 1). The amount of restriction enzyme required for complete digestion is comparable with DNA purified by other methods. In addition to blood, DNA has been isolated from a variety of cell lines and tissues. The DNA is suitable for a number of other enzymatic modifications such as kinasing, ligation, and amplification by PCR. Utilization of this procedure allows the isolation of DNA from a large number of samples in less than 4 hours.

Missense variants in AIMP1 gene are implicated in autosomal recessive intellectual disability without neurodegeneration

“…Genomic DNA was extracted from peripheral blood of affected and unaffected members of family PMKR60 and the patients and their mother of family M105 using standard procedures. 19 …”

A compound heterozygous mutation in DPAGT1 results in a congenital disorder of glycosylation with a relatively mild phenotype

“…DNA isolation was carried out by using the standard protocols. 9 Genotyping and copy number variation analysis…”

Tumor necrosis factor gene polymorphisms in patients with cirrhosis from chronic hepatitis C virus infection

“…DNA extraction and PCR-based genotyping of TNF␣, LT␣, TNFc, and TNFR1 allelic variants Genomic DNA was isolated from whole buffy coats of participants using standard inorganic salt-out procedures 30,31 High resolution polymerase chain reaction with sequence-specific primers (PCR-SSP) was used to define polymorphisms in the TNF␣ promoter at positions −238 and −308, and at LT␣ position 26. 18 Reference samples whose TNF␣ and LT␣ alleles had been established by DNA sequencing and restriction enzyme digestion were used to validate the SSP-based genotyping strategy.…”