Table 1.
Experimental design of DNA extraction for PCR and on-chip-electrophoresis.
Table 2.
Experimental design of DNA extraction for real-time PCR
Table 3.
Annealing temperatures.
Figure 1.
Amplification results using the proposed DNA extraction method (on-chip- electrophoresis results).
The size of PCR products are 79–115 bp (CSRM60), 100–124 bp (INRA035). Left to right: L, ladder; 1, sample washing water control (CSRM60); 2, extraction solution (without hair shaft) control (CSRM60); 3, ddH2O control (CSRM60); 4, DNA from hair shafts of pure bred Luxi cattle (CSRM60); 5, DNA from liver of beef cattle (CSRM60); 6, sample washing water control (INRA035); 7, extraction solution (without hair shaft) control (INRA035); 8, ddH2O control (INRA035); 9, DNA from hair shafts of pure bred Luxi cattle (INRA035); 10, DNA from liver of beef cattle (INRA035).
Figure 2.
Comparison of amplification results of DNA extracted from hair shafts using the proposed method and that extracted from liver using commercial Genomic DNA Purification Kit (on-chip-electrophoresis results).
The above panel is INRA035 comparison result, sample 6 is amplification result of hair shaft DNA from pure bred Luxi cattle and sample 3 is amplification result of liver DNA from beef cattle; The below panel is CSRM60 comparison result, sample 2 is amplification result of hair shaft DNA from pure bred Luxi cattle and sample 11 is amplification result of liver DNA from beef cattle.
Figure 3.
Effects on the efficiency of real-time PCR of sample amount used for extraction and input template volume.
(A) Second round amplification result (CT value of ETH225). Six sample amount groups (5 mg, 2 mg, 1 mg, 0.5 mg, 0.2 mg, 0.1 mg) and six template volume groups (different template volumes in first round, but same template volume (2 µl) in second round) of Diao™ enzymatic laundry powder. (B) First round amplification result (CT value of HAUT27). Six sample amount groups (5 mg, 2 mg, 1 mg, 0.5 mg, 0.2 mg, 0.1 mg) and six template volume groups of Diao™ enzymatic laundry powder. Because of amplification inhibition factors, no CT value of 5 µl, 2 µl, 1 µl template group and some of 0.5 µl and 0.2 µl template groups were obtained (“undetermined”). Negative controls are not shown in this figure; their CT values were undetermined too.
Figure 4.
Negative correlation between PCR efficiency and the amount of input template.
The data of all three enzymatic laundry powder groups (each detergent group has six sample amount groups: 5 mg, 2 mg, 1 mg, 0.5 mg, 0.2 mg, 0.1 mg; and each sample amount group has six template volume groups: 5 µl, 2 µl, 1 µl, 0.5 µl, 0.2 µl, 0.1 µl) were adopted (target locus ETH225, second round), Spearman's rho = 0.810, P<0.001, n = 107 (one sample has no CT value and has been excluded by the SPSS). 90 of 107 points were in circular ROI box (yellow oval area), mean CT value is 8.7, SD = 4.1; and median CT value is 6.0.
Table 4.
DNA yield of extraction with different enzymatic laundry powder and sample amounts.
Table 5.
Results after two rounds of Real-time PCR using different enzymatic laundry powder, sample amounts and template volume in the first round PCR.
Table 6.
Comparison of different DNA extraction methods.
Figure 5.
Photographic image of the extraction solution with different sample amounts.