Sequencing Service Based on PCR-Amplified cpDNA

Chloroplast DNA (cpDNA) sequence variation is currently widely used to study interspecific relationships between angiosperms and other plants, but intraspecific studies are difficult. In addition, restriction fragment length polymorphisms also occur at the plant species level, with the highest mutation frequency in noncoding regions. Therefore, the resolution of cpDNA can be improved by polymerase chain reaction (PCR) amplification and direct sequencing of these noncoding regions for evolutionary studies and identification of intraspecific genetic markers. PCR, as an important molecular technique, has applications in almost all fields of biomedical and biotechnological sciences. PCR can generate many copies of a specific part of DNA, allowing for more detailed manipulation and analysis of DNA fragments. Scientists have now designed primers for the tRNA gene sequences of many plants and obtained sequences by sequencing PCR-amplified long cpDNA fragments, providing opportunities to study the population and evolutionary biology of various plant species.

Fig.1. Illustration of the two-step PCR approach. (Cruaud P, et al., 2017)

Services

The availability of chloroplast genomes in certain species such as conifers is still limited due to the high content of secondary metabolites and the difficulty in isolating chloroplasts. Therefore, our scientists are committed to developing advanced technologies to meet this challenge. We have successfully designed primers for amplifying and sequencing DNA fragments from chloroplast genes and noncoding regions to help you build a variety of plant trees of life for molecular phylogenetic studies. Over the years, the team at Lifeasible has accumulated extensive experience in sequencing chloroplast genes. Here, our scientists are dedicated to providing sequencing service based on PCR-amplified cpDNA, using Sanger and Illumina paired-end sequencing technologies to sequence PCR-amplified long cpDNA fragments to obtain the complete chloroplast genome sequence. Through our advanced sequencing platform, we can help you complete the DNA amplification and sequencing of different plant species. The flow of sequencing service based on PCR-amplified cpDNA is as follows:

(1) Amplifing the entire chloroplast genome using PCR and designed primers to obtain cpDNA.
(2) cpDNA was sequenced according to Sanger and Illumina sequencing methods, and the sequenced genome was annotated.
(3) Detecting and validating variants (SNPs).
(4) Sequencing accuracy estimation.
(5) Sequencing alignment and phylogenetic analysis to mine more information.
(6) Providing a complete data analysis report.

Competitive Advantages

• PCR amplification combined with sequencing can eliminate the step of purifying cpDNA.
• Primers with high specificity are required and are difficult to identify without reference chloroplast genomes of related species.
• Widely applicable to secondary metabolites to isolate the large nuclear genome of chloroplasts.
• More favorable for variant (SNP and indel) prediction.
• Great advantages in experimental time, analysis time and cost, it only takes 2-3 days with PCR + Sanger.
• The experimental time of PCR+Sanger was mainly used for PCR amplification of cpDNA fragments.
• In addition to providing standard bioinformatics services, also provide personalized bioinformatics services.

Lifeasible can meet the needs of customers on time and on budget through a wide range of chloroplast DNA sequencing  strategies. Our aim is to be customer-centric and to provide the highest quality service to customers around the world. Our skilled and dedicated scientific researchers ensure that the most appropriate methods and techniques are selected for each specialized chloroplast project. Our customer service representatives are enthusiastic and trustworthy 24 hours a day, 7 days a week. If you are interested in our services, please feel free to contact us for more information or a detailed discussion.

References

1. Hamilton K, Barfoot J, Crawford K E, et al. (2006) Amplification of chloroplast DNA using the polymerase chain reaction (PCR): a practical activity for secondary school students[J]. Journal of Biological Education. 40(4): 172-177.
2. Cruaud P, Rasplus J Y, Rodriguez L J, et al. (2017) High-throughput sequencing of multiple amplicons for barcoding and integrative taxonomy[J]. Scientific reports. 7(1): 1-12.