Abstract
ABSTRACT INTRODUCTION: Deoxyribonucleic acid (DNA) is the raw material for genetic studies, and therefore laboratory techniques are developed to obtain it with appropriate concentration and integrity. OBJECTIVE: To compare two methods of DNA extraction regarding sample concentration and integrity. METHODS: DNA was extracted from the tail end (2 mm long) of mice, stored at -20oC. The proteinase K method (PKM) and the Kappa Express Extract® kit were used for extraction. The concentrations and ratios 260/280 and 260/230 were determined by spectrophotometry. DNA integrity was checked on 2% agarose gel with ethidium bromide. For the final test of the extracted samples, a multiplex polymerase chain reaction (PCR) was performed, with primers for the Large gene. RESULTS: Samples extracted by the PKM presented mean concentration value of 59.4 ± 18.5 ng/µl (260/280 = 1.74 ± 0.04 and 260/230 + 1.85 ± 0.14) and the samples extracted by the commercial kit presented mean concentration value of 178.8 ± 42.0 ng/µl (260/280 = 1.09 ± 0.04 and 260/230 = 0.62 ± 0.66). PCR amplified the Large gene in the DNA extracted, regardless of the methodology used. CONCLUSION: Both methodologies studied can be used, and the PKM is cheap but a time-consuming method, while the commercial kit is more expensive, however DNA extraction is faster.
Highlights
Deoxyribonucleic acid (DNA) is the raw material for genetic studies, and laboratory techniques are developed to obtain it with appropriate concentration and integrity
Samples extracted by the proteinase K method (PKM) showed a mean concentration of 59.4 ± 18.5 ng/μl with 260/280 and 260/230 ratios of 1.74 ± 0.04 and 1.85 ± 0.14, respectively (Table 2 and Figure 1)
(0.04 ng/μl to 0.08 ng/μl); they observed that high concentrations of proteinase K in the reaction increase DNA degradation, that is, negatively influence its integrity. These results are similar to those in this study found in the samples obtained with the commercial extraction kit
Summary
Deoxyribonucleic acid (DNA) is the raw material for genetic studies, and laboratory techniques are developed to obtain it with appropriate concentration and integrity. Objective: To compare two methods of DNA extraction regarding sample concentration and integrity. Methods: DNA was extracted from the tail end (2 mm long) of mice, stored at -20°C. The proteinase K method (PKM) and the Kappa Express Extract® kit were used for extraction. For the final test of the extracted samples, a multiplex polymerase chain reaction (PCR) was performed, with primers for the Large gene. PCR amplified the Large gene in the DNA extracted, regardless of the methodology used. Conclusion: Both methodologies studied can be used, and the PKM is cheap but a time-consuming method, while the commercial kit is more expensive, DNA extraction is faster
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