DNA Extraction from Dry Rhizomes for DNA Barcoding. Development of a Cost-Effective Method

Table of Contents

• CHAPTER 01 INTRODUCTION
• CHAPTER 02 LITERATURE REVIEW
• 2.1 Zingiberaceae Family
• 2.1.1 Alpinia galanga – Maha aratta
• 2.1.2 Alpinia calcarata- Heen aratta
• 2.1.3 Alpinia purpurata - Red ginger
• 2.1.4 Alpinia malaccensis - Ran kinihiriya
• 2.1.5 Alpinia abundiflora
• 2.1.6 Hedychium coronarium - Ela mal
• 2.1.7 Hedychium coccineum - Kaula ala
• 2.2 Plant Sample Collection and Storage for DNA Extraction
• 2.2.1 Analysis of Fresh Sample
• 2.2.2 Analysis of Dry Sample
• 2.3 DNA Extraction Methods for Plants with High Content of Secondary Metabolites
• 2.3.1 Issues with Secondary Metabolites in DNA extraction
• 2.3.2 Plant Mini Kit Protocol
• 2.3.3 CTAB Extraction Method
• 2.3.4 Modifications of CTAB Method
• 2.4 DNA Quantification
• 2.4.1 Gel Electrophoresis
• 2.5 Purity Testing of Extracted DNA
• 2.5.1 Restriction Digestion
• 2.5.2 UV Spectrophotometer
• CHAPTER 03 MATERIALS AND METHOD
• CHAPTER 04 RESULTS AND DISCUSSIONS
• CHAPTER 05 CONCLUSION

Objectives and Key Themes

The main objective of this research was to develop a cost-effective method for DNA extraction from dry rhizomes of Alpinia calcarata (Heen aratta) suitable for DNA barcoding. This is crucial for accurate identification of the plant, preventing adulteration with related species and ensuring the quality of Ayurvedic medicine.

• Development of a low-cost DNA extraction method.
• Evaluation of different DNA extraction methods.
• Assessment of DNA quality and quantity.
• Cost analysis of the developed method.
• Application of DNA barcoding for species identification.

Chapter Summaries

CHAPTER 01 INTRODUCTION: This chapter introduces the research topic, focusing on the importance of accurate identification of Alpinia calcarata (Heen aratta) in the context of its use in Ayurvedic medicine. The chapter highlights the challenges of using morphological characterization for identification due to similarities with related species, leading to potential adulteration and reduced medicinal value. It establishes the need for a reliable and cost-effective DNA extraction method for DNA barcoding as a solution to this problem, setting the stage for the subsequent research presented in the dissertation.

CHAPTER 02 LITERATURE REVIEW: This chapter provides a comprehensive overview of existing literature related to the research. It delves into the Zingiberaceae family, focusing on the different species of Alpinia, including A. calcarata, and their morphological similarities and differences. This section critically examines methods for plant sample collection and storage for DNA extraction, emphasizing the challenges associated with the analysis of dry samples, especially those high in secondary metabolites. The review also explores different DNA extraction methods focusing on their strengths and weaknesses concerning cost-effectiveness and suitability for plants rich in secondary metabolites. It concludes by summarizing common methods for DNA quantification and purity testing.

CHAPTER 03 MATERIALS AND METHOD: This chapter details the materials and methodology used in the research. It describes the location and collection of plant materials, outlines the equipment used, and provides a step-by-step description of the three DNA extraction methods employed (SDS, 4x CTAB, and CTAB series). The chapter also outlines the methods used for DNA quantification (using UV spectrophotometer and known DNA samples), purity testing (restriction digestion), and PCR amplification. This section provides the necessary information to understand and replicate the experiments conducted.

CHAPTER 04 RESULTS AND DISCUSSIONS: This chapter presents and analyzes the results obtained from the experiments. It discusses the outcomes of each DNA extraction method, including the quantification and purity of extracted DNA. The results of restriction digestion reaction and PCR amplification, indicating the suitability of the extracted DNA for downstream applications, are discussed in detail. A cost analysis of the three methods is presented, highlighting the cost-effectiveness of the selected method. The chapter concludes by discussing the implications of the findings for the accurate identification of A. calcarata and the improvement of quality control in Ayurvedic medicine production.

Keywords

Cost-effective method, Heen aratta, Alpinia calcarata, DNA extraction, DNA barcoding, Restriction digestion, PCR amplification, Ayurvedic medicine, species identification, secondary metabolites, CTAB method, UV spectrophotometry.

Frequently Asked Questions: A Comprehensive Language Preview

What is the main objective of this research?

The primary goal is to develop a cost-effective DNA extraction method from dry rhizomes of Alpinia calcarata (Heen aratta) suitable for DNA barcoding. This is vital for accurate plant identification, preventing adulteration with similar species, and ensuring Ayurvedic medicine quality.

What are the key themes explored in this research?

The research focuses on developing a low-cost DNA extraction method, evaluating different extraction techniques, assessing DNA quality and quantity, conducting a cost analysis of the developed method, and applying DNA barcoding for species identification.

Which plants are discussed in the literature review?

The literature review examines the Zingiberaceae family, specifically focusing on various Alpinia species, including A. calcarata (Heen aratta), A. galanga (Maha aratta), A. purpurata (Red ginger), A. malaccensis (Ran kinihiriya), A. abundiflora, Hedychium coronarium (Ela mal), and Hedychium coccineum (Kaula ala). It also discusses methods for plant sample collection and storage, focusing on challenges with dry samples high in secondary metabolites.

What DNA extraction methods are compared?

The research compares several DNA extraction methods, including a plant mini kit protocol, the CTAB extraction method, and modifications of the CTAB method. The effectiveness and cost-effectiveness of each method are evaluated.

How is DNA quality and quantity assessed?

DNA quality and quantity are assessed using methods such as gel electrophoresis, UV spectrophotometry, and restriction digestion. The suitability of the extracted DNA for PCR amplification is also evaluated.

What are the chapter summaries?

Chapter 1 (Introduction) introduces the research topic, highlighting the importance of accurate Alpinia calcarata identification in Ayurvedic medicine and the challenges of using morphological methods. Chapter 2 (Literature Review) provides a comprehensive overview of relevant literature, focusing on the Zingiberaceae family, sample collection and storage, DNA extraction methods, and quality assessment techniques. Chapter 3 (Materials and Methods) details the materials and methodology employed in the research, including the DNA extraction methods used (SDS, 4x CTAB, and CTAB series), DNA quantification, purity testing, and PCR amplification. Chapter 4 (Results and Discussion) presents and analyzes the results of the experiments, discussing the effectiveness of each DNA extraction method, cost analysis, and implications for Ayurvedic medicine quality control. Chapter 5 (Conclusion) summarizes the findings and their significance.

What are the key words associated with this research?

Key words include: Cost-effective method, Heen aratta, Alpinia calcarata, DNA extraction, DNA barcoding, Restriction digestion, PCR amplification, Ayurvedic medicine, species identification, secondary metabolites, CTAB method, UV spectrophotometry.