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Novel method streamlines blood DNA purification for PCR applications
Sylvia Norman Jeff Braman
Stratagene
Using the new DNA Stat blood kit, it is easy to isolate PCR-quality human genomic DNA from whole blood with a uniquely designed spin-cup. Genomic DNA is quickly purified from 25 l to 200 l of whole blood with fewer steps and less handling compared to other methods; the resulting DNA is approximately 40 kb. The PCR targets tested and amplified ranged from 250 bp to 5.2 kb. To demonstrate that all human chromosomes were present in DNA isolated from human blood, we amplified simple sequence repeat markers. This novel isolation procedure was also shown to be successful in a clinical reference laboratory setting.
Genomic DNA isolated from whole blood has a broad range of PCR analysis applications that include typing HLA class I and II alleles,1,2,3 screening neonates,4 detecting tumor cells,5 and studying allelic discrimination.6,7,8 Commercially available genomic DNA isolation kitswe designed a simple method that does not require multiple transfer steps, the use of proteases with their attendant incubations, phenol-chloroform extractions, alcohol precipitation, or additional incubations above room temperature.
DNA Stat Blood Kit Protocol
Fig.1
The key to the DNA Stat blood kit is the unique design of the spin cups, which preferentially bind white blood cells (WBC) from a whole blood sampleol (Figure 1), a hypotonic solution is added to a microspin cup seated in a 2-ml collection tube. A whole blood sample (25 l to 200 l) is then added to this solution, and the red blood cells (RBC) are lysed. Centrifugation removes RBC and serum protein contaminants while the WBC are retained. The WBC are lysed by adding a chaotropic salt solution, and the released genomic DNA is bound to the DNA Stat spin cup. Residual contaminants are removed with two wash steps, and the genomic DNA is then eluted in a small volume of buffer. The resulting DNA is high molecular weight and ready for use as a template in PCR reactions. Each prep yields enough DNA for up to 50 PCR reactions with 1 ml to 5 ml of DNA, providing sufficient template for most reactions.
When comparing contemporary protocols with Stratagenes kit (Table 1), the DNA Stat method demonstrates several advantages: no pelleting of WBC before lysis, no protease digestion, and room temperature incubations. A single microspin cup is all that is necessary to carry out the isolation, one simple protocol is used for a range of blood volumes, and the entire isolation takes only 40 minutes.
Convenient DNA Isolation from Whole Blood
Fig.2
The DNA Stat kit can be used to isolate DNA from a range of whole blood volumes without making any changes to the protocol. Other commercially available blood DNA isolation kits are optimized for a specific blood volume, and any change in starting volume requires protocol modification. All isolations were performed under identical conditions using the genomic DNA isolation procedure described in the kits manual (Figure 2). PCR analysis was performed using 1 ml of the isolated genomic DNA as template and primers specific for 1-antitrypsin (a1-AT). The results demonstrate that 1 ml of genomic DNA template isolated from a range of blood volumes is sufficient for amplification of the expected 980-bp PCR product.
Amplification of Various PCR Target Sizes
Fig.3
Genomic DNA isolated from blood using the DNA Stat kit is high molecular weight (approximately 40 kb in size) (Figure 3). To further test the integrity of the isolated genomic DNA, 1-ml aliquots of a 50-ml prep were used as templates for amplifying a range of PCR target sizes. We used primers specific to aldolase B (Aldo: 250 bp), T-cell receptor b-chain gene (TCRb: 500 bp), and a1-antitrypsin (a1-AT: two primer sets amplifying 980-bp and 5.2K-bp PCR targets) in PCR reactions with 1 ml al of genomic DNA. Genomic DNA isolated using the DNA Stat kit is suitable for amplifying PCR targets ranging in size from 250 bp to 5.2 kb (Figure 4).
Fig.4
We also assessed genomic DNA quality by amplifying PCR targets on each of the human chromosomes. Primers designed to amplify targets containing simple sequence repeats on chromosomes 1 through 22 plus X and Y were used for this analysis (Human MapPairs primers, Research Genetics: TCRb primers were used for chromosome 7). The resulting PCR products ranged in size from 202 bp to 500 bp. From 100 ml of whole blood, 1 ml of DNA was used as a template for PCR reactions. The expected amplification products for each chromosome were obtained, indicating that the genomic DNA isolated using the DNA Stat kit contains a complete representation of all human chromosomes (data not shown).
Isolated Genomic DNA for Reference Laboratory Analysis
The DNA Stat kit was used in a reference laboratory setting (Cenetron, Inc.) to isolate genomic DNA from patient whole blood samples. The resulting DNA was used as template for quantitative PCR analysis with primers specific to Factor V. Results from PCR reactions using 5 ml of template DNA isolated from six individual whole blood samples showed that each reaction produced the expected 107-bp PCR product and, therefore, the isolated genomic DNA is suitable for use in clinical reference laboratory assays (data not shown).
Conclusions
The quick and easy-to-use DNA Stat is the choice for isolating PCR-ready genomic DNA from whole blood. The isolated DNA is high molecular weight (40 kb) and an excellent template for PCR analysis. Because of the kits unique spin-cup design, there is no need to remove RBC or pellet WBC before beginning the procedure. Additionally, there is no need for alcohol precipitation, no need to digest with proteases, and no need to alter the protocol with a 25-l to 200-l blood volume. Furthermore, all incubations are carried out at room temperature, all steps are performed in a single microspin cup, and all isolations are easily completed in 40 minutes. An aliquot of 1 ml to 5 ml of the isolated genomic DNA is sufficient for most PCR analyses including clinical reference laboratory assays.
REFERENCES
1. Carter, A.S., et al. (1999) Blood 94: 1471-1477.
2. Ramseier-Zanone, R., et al. (1999) Transplantation 67: 1336-1341.
3. Mwenda, J., et al. (1999) Cell Mol. Biol. 45: 1249-1256.
4. Dobrowolski, S.F., et al. (1999) Acta Paediatr. Suppl. 88: 61-64.
5. Debled, M., et al. (1999) Intl. J. Cancer 84: 558-561.
6. Potzsch, C., Schaefer, H.E., and Lubbert, M. (1999) Am. .J Hematol.
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7. Kristensen, T., et al. (1998) Biotechniques 25: 832-835.
8. Larsen, L.A., et al. (1997) Hum. Genet. 100: 564-568.
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