A DNA extraction method using Chelex 100 is widely used for bacteria, Chlamydomonas, and animal cell lines, but only rarely for plant materials due to the need for additional time-consuming and tedious steps. We have modified the Chelex 100 protocol and successfully developed a rapid and simple meth...
A DNA extraction method using Chelex 100 is widely used for bacteria, Chlamydomonas, and animal cell lines, but only rarely for plant materials due to the need for additional time-consuming and tedious steps. We have modified the Chelex 100 protocol and successfully developed a rapid and simple method of DNA extraction for efficient PCR-based detection of transgenes from a variety of transgenic plant and algal species. Our protocol consists of homogenizing plant tissue with a pestle, boiling the homogenized tissue in a microfuge tube with 5% Chelex 100 for 5 min, and centrifuging the boiled mixture. The supernatant, which is used for PCR analysis, was able to successfully amplify transgenes in transgenic tobacco, tomato, potato, Arabidopsis, rice, strawberry, Spirodela polyrhiza, Chlamydomonas, and Porphyra tenera. The entire DNA extraction procedure requires <15 min and is therefore comparable to that used for bacteria, Chlamydomonas, and animal cell lines.
A DNA extraction method using Chelex 100 is widely used for bacteria, Chlamydomonas, and animal cell lines, but only rarely for plant materials due to the need for additional time-consuming and tedious steps. We have modified the Chelex 100 protocol and successfully developed a rapid and simple method of DNA extraction for efficient PCR-based detection of transgenes from a variety of transgenic plant and algal species. Our protocol consists of homogenizing plant tissue with a pestle, boiling the homogenized tissue in a microfuge tube with 5% Chelex 100 for 5 min, and centrifuging the boiled mixture. The supernatant, which is used for PCR analysis, was able to successfully amplify transgenes in transgenic tobacco, tomato, potato, Arabidopsis, rice, strawberry, Spirodela polyrhiza, Chlamydomonas, and Porphyra tenera. The entire DNA extraction procedure requires <15 min and is therefore comparable to that used for bacteria, Chlamydomonas, and animal cell lines.
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제안 방법
DNA, primers, Taq polymerase, and reaction buffers were mixed for PCR cycling (GeneAmp PCR system 9700; Applied Biosystems, Foster City, CA) and amplified under the following cycling conditions: one cycle at 94℃ for 5 min, followed by 30–40 cycles of 94℃ for 30 s, 52–58℃ for 30 s, and 72℃ for 1–2 min, and terminated by a final 5-min extension at 72℃. The following primer pairs were used to amplify the target genes: TBP19-F (50 -CGCCATGGAACGAGCTATAC A-30 ) and TBP19-R (50 -CCTCTAGAAGGTCTCAGTACC T-30 ) amplified a 0.5-kb TBP19 fragment; NPTIIF (50 -G AGGCTATTCGGCTATGACTG-30 ) and NPTIIR (50 -ATC GGGAGCGGCGATACCGTA-30 ) amplified a 0.7-kb nptII fragment; btbP-5 (50 -CCACTCGAGCTTGTGATCGCA CT-30 ) and Aph7R2 (50 -TTCCGGTCGGTCGTGCCGTCC AT-30 ) amplified a 0.6-kb aph7 fragment; PtHSP70R3 (50 -A CAGGAGCCGACGCGTGCCA-30 ) and PtHSP70P1KF (50 -TTGGATCCGAACCTGCCCCCGGGT-30 ) amplified a 1.1-kb heat shock protein (HSP 70) fragment from P. tenera; PtBtub (31 50 -CTCCGASACSAGGTCRTTCA T-30 ) and PtBtub51 (50 -AACAACTGGGCYAAGGGSC A-30 ) amplified a 1-kb beta tubulin (bTub) fragment from P. tenera.
To develop a universal plant DNA extraction protocol, we tested the applicability of our modified Chelex 100 method with DNA from various species. In this study, we used our method to extract DNA from tomato tissues cultured in vitro and grown in a growth chamber; the DNA was subsequently tested by PCR amplification. We found that the transgene from the DNA of transgenic tomato plants was successfully amplified (Fig.
성능/효과
Homogenization of plant tissue is considered to be critical for successful PCR amplification after rapid DNA extraction. To test this assumption, we evaluated PCR efficiency after DNA extraction without the homogenization step and found that although the PCR did not always amplify transgenes without the homogenization step, the amplification itself appeared to be successful if the boiling time was extended or the number of PCR cycles was increased. Furthermore, at least a 5-min incubation in boiling water was required to produce rapid and consistent PCR amplification.
This method was evaluated using a diverse range of plant species including potato, Arabidopsis, rice, strawberry, S. polyrhiza (hydrophyte), C. reinhardtii (green algae), and Porphyra tenera (red alga), and the genes of interest were successfully amplified from all species tested (Fig. 2 ). Although the amplification of some DNA samples produced only faint PCR bands following electrophoresis in an agarose gel, increasing the number of PCR cycles or the quantity of the DNA template produced stronger bands, whereas the same conditions still produced no signal from the DNA of wild-type plants lacking the transgene.
참고문헌 (9)
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