Chloroplast DNA Extraction

Chloroplast DNA (cpDNA) from green plants was the target of early genome sequencing projects due to its small size. At least 200 plant species have complete cpDNA sequenced, providing valuable genetic information for plant evolution and function studies. Lifeasible's expertise in chloroplast engineering has allowed us to isolate high-quality cpDNA with high yield and reliable purity from all herbaceous and woody plants. If you have any special requirements about our chloroplast cpDNA extraction service, please feel free to contact us.

Chloroplast DNA (cpDNA)

Chloroplasts are plant organelles containing circular DNA that contain 110-130 genes ranging in size from 120-160 kilobases (kb). As a semi-autonomous organelle, the chloroplast possesses its own independent genome and complete transcription and translation machinery to express its genetic information. In addition, cpDNA is usually inherited maternally and does not require genetic recombination. Studies have shown that cpDNAs provide rich information for plant phylogeny, molecular ecology, comparative genomics, population genetics, and evolution by using the entire chloroplast genome sequence. The complete cpDNA of at least 200 plants has been sequenced. And with the application of next-generation sequencing technology in plastid genome sequencing projects, the number of fully sequenced chloroplast genomes has increased dramatically. Scientists use sucrose density gradient centrifugation, high-salt buffer, and Percoll gradient centrifugation for isolation of cpDNA from fresh plant material, but are susceptible to contamination. Therefore, the extraction of high-quality cpDNA from certain species(such as millet, rice and other grass crops) has been a challenge for researchers.

Fig. 1. Flowchart of chloroplast DNA isolation using liquid nitrogen coupled with sucrose gradient centrifugation. (Takamatsu T, et al., 2018)

Services

Many areas of chloroplast research require methods that can assess cpDNA quality and quantity, and obtaining high-quality cpDNA is a prerequisite for chloroplast genome research. A protocol that attempts to isolate chloroplasts directly prior to cpDNA isolation will improve the quality of cpDNA sequencing. Lifeasible has successfully developed methods for the isolation of sequencing-quality intact cpDNA from all herbaceous and woody plants. Based on the advantages and disadvantages of each method, we will provide the best cpDNA isolation solution for your project.

• Liquid nitrogen combined with sucrose density gradient centrifugation.
• High salt buffer method.
• Percoll gradient centrifugation.
• Polymerase chain reaction (PCR) amplified cpDNA fragments from total genomic DNA.

Because the leaves of some grass crops are thin and fibrous, the content of chloroplasts in mesophyll cells is low, and the abundant wax, cuticle and silica on the surface of the leaves also make it difficult for cells to break down and release chloroplasts. Therefore, we improved the extraction protocol based on the traditional cpDNA extraction method to improve the quality of cpDNA isolation. The program flow is as follows:

(1) Isolation of chloroplasts from leaf tissue: use a scalpel blade to cut leaves into small pieces to obtain intact chloroplasts.

(2) Separating chloroplasts from other components: Choose an appropriate centrifugation speed and density gradient to separate chloroplasts from cellular debris and mitochondrial DNA, and collect crude chloroplast pellets and chloroplasts based on differences in sedimentation rates between mitochondria.

(3) Purifing cpDNA.

Competitive Advantages

• A fast and efficient cpDNA extraction procedure.
• Allowing the extraction of enriched cpDNA to study plastid genomes in a more cost-effective and time-efficient manner, and greatly increases sequence throughput.
• Protecting chloroplast integrity and significantly improves cpDNA yield and quality.
• Isolated cpDNA has been widely used to study genome-wide phylogeny and ecology, transcriptomics, and complete plastid proteome characterization.

References

1. Takamatsu T, Baslam M, Inomata T, et al. (2018) Optimized method of extracting rice chloroplast DNA for high-quality plastome resequencing and de novo assembly[J]. Frontiers in Plant Science. 9: 266.
2. Liu D, Cui Y, Li S, et al. (2019) A new chloroplast DNA extraction protocol significantly improves the chloroplast genome sequence quality of foxtail millet (Setaria italica (L.) P. Beauv.)[J]. Scientific reports. 9(1): 1-9.