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An improved chloroplast DNA extraction procedure for whole plastid genome sequencing.

Shi C, Hu N, Huang H, Gao J, Zhao YJ, Gao LZ - PLoS ONE (2012)

Bottom Line: Both of them failed to obtain properly defined cpDNA bands.The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly.The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.

View Article: PubMed Central - PubMed

Affiliation: Plant Germplasm and Genomics Center, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.

ABSTRACT

Background: Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs.

Methodology/principal findings: We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40-50% cpDNA purity is achieved with our method.

Conclusion: Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.

Show MeSH
HindIII restriction enzyme digestion of isolated cpDNAs from the three plant species.a) O. brachyantha, b) L. japonica, and c) P. utihis. The DNA bands were shown on a 0.8% agarose gel, and DNA was isolated with the improved high salt method; M indicates 1 kbp DNA ladder.
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pone-0031468-g003: HindIII restriction enzyme digestion of isolated cpDNAs from the three plant species.a) O. brachyantha, b) L. japonica, and c) P. utihis. The DNA bands were shown on a 0.8% agarose gel, and DNA was isolated with the improved high salt method; M indicates 1 kbp DNA ladder.

Mentions: As a modification of the sucrose gradient centrifugation, the high salt method significantly simplified the cpDNA isolation process. By using this method, our first effort to isolate the cpDNAs also seems successful, as it can get a relatively clearly defined DNA band. When we increased the amount of fresh leaves, however, a positive correlation between increased DNA yield and the possibility of DNA degradation was found, indicating that there is more contamination of nuclear DNAs (Figure 2). The observation suggests that this method may not be suitable to isolate cpDNAs with high purity. As an alternative approach in chloroplast isolation, four to six volumes (v/w) cold isolation buffer (in the original protocol) may not be enough to homogenate the fresh leaves (e.g., 20 g fresh leaves with 100 ml isolation buffer). Therefore, we increased the amount of isolation buffer from 5 to 20 volumes of fresh leaves (e.g., 20 g fresh leaves with 400 ml buffer A in our protocol) in the subsequent experiment. Even when 50 g fresh leaves were used, a well-defined cpDNA band can be observed (Figure 2), suggesting that the modification led to a successful isolation of the cpDNAs. It is likely that about 20 g leaves may be more optimal as it could include less contaminating nuclear DNA. Furthermore, two additional centrifugation steps (200 g 20 min and 3500 g 20 min, separately) were used to discard the cell debris and collect chloroplast pellet. To decrease the nuclear DNA contamination that adheres to the outer chloroplast membrane, we also incorporated extra steps to wash the chloroplast pellet with buffer B, further increasing the purity of isolated cpDNAs. Last but not least, chloroplasts were lysed using SDS and Proteinase K instead of cetyltrimethylammonium bromide (CTAB) followed by phenol/chloroform extraction. The final isolated cpDNAs were digested with HindIII and the result was visualized on a 0.8% agarose gel (Figure 3). Among these modifications, incorporated gradual centrifugation steps were of the most importance, because they are able to increase the cpDNA purity by separating the chloroplasts from cell debris. If larger amounts of starting materials (e.g., 50 g fresh leaves) were used, it is necessary to add a second centrifugation step at 200 g.


An improved chloroplast DNA extraction procedure for whole plastid genome sequencing.

Shi C, Hu N, Huang H, Gao J, Zhao YJ, Gao LZ - PLoS ONE (2012)

HindIII restriction enzyme digestion of isolated cpDNAs from the three plant species.a) O. brachyantha, b) L. japonica, and c) P. utihis. The DNA bands were shown on a 0.8% agarose gel, and DNA was isolated with the improved high salt method; M indicates 1 kbp DNA ladder.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285163&req=5

pone-0031468-g003: HindIII restriction enzyme digestion of isolated cpDNAs from the three plant species.a) O. brachyantha, b) L. japonica, and c) P. utihis. The DNA bands were shown on a 0.8% agarose gel, and DNA was isolated with the improved high salt method; M indicates 1 kbp DNA ladder.
Mentions: As a modification of the sucrose gradient centrifugation, the high salt method significantly simplified the cpDNA isolation process. By using this method, our first effort to isolate the cpDNAs also seems successful, as it can get a relatively clearly defined DNA band. When we increased the amount of fresh leaves, however, a positive correlation between increased DNA yield and the possibility of DNA degradation was found, indicating that there is more contamination of nuclear DNAs (Figure 2). The observation suggests that this method may not be suitable to isolate cpDNAs with high purity. As an alternative approach in chloroplast isolation, four to six volumes (v/w) cold isolation buffer (in the original protocol) may not be enough to homogenate the fresh leaves (e.g., 20 g fresh leaves with 100 ml isolation buffer). Therefore, we increased the amount of isolation buffer from 5 to 20 volumes of fresh leaves (e.g., 20 g fresh leaves with 400 ml buffer A in our protocol) in the subsequent experiment. Even when 50 g fresh leaves were used, a well-defined cpDNA band can be observed (Figure 2), suggesting that the modification led to a successful isolation of the cpDNAs. It is likely that about 20 g leaves may be more optimal as it could include less contaminating nuclear DNA. Furthermore, two additional centrifugation steps (200 g 20 min and 3500 g 20 min, separately) were used to discard the cell debris and collect chloroplast pellet. To decrease the nuclear DNA contamination that adheres to the outer chloroplast membrane, we also incorporated extra steps to wash the chloroplast pellet with buffer B, further increasing the purity of isolated cpDNAs. Last but not least, chloroplasts were lysed using SDS and Proteinase K instead of cetyltrimethylammonium bromide (CTAB) followed by phenol/chloroform extraction. The final isolated cpDNAs were digested with HindIII and the result was visualized on a 0.8% agarose gel (Figure 3). Among these modifications, incorporated gradual centrifugation steps were of the most importance, because they are able to increase the cpDNA purity by separating the chloroplasts from cell debris. If larger amounts of starting materials (e.g., 50 g fresh leaves) were used, it is necessary to add a second centrifugation step at 200 g.

Bottom Line: Both of them failed to obtain properly defined cpDNA bands.The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly.The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.

View Article: PubMed Central - PubMed

Affiliation: Plant Germplasm and Genomics Center, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.

ABSTRACT

Background: Chloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs.

Methodology/principal findings: We first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40-50% cpDNA purity is achieved with our method.

Conclusion: Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.

Show MeSH