This research was done to develop a low-cost method for DNA extraction from the dry rhizomes of Heen aratta. DNA extraction is very hard from dry rhizomes because contain of high polyphenolic content. Three modified method methods were followed to extract the DNA from dry rhizomes, SDS, 4x CTAB, and CTAB series methods.
Alpinia calcarata (heen aratta) is widely used as ayurvedic medicine in Sri Lanka. Medicinal uses are treatments for indigestion, impurities of blood, throat inflammation and voice improvement, cough, respiratory ailments, bronchitis, asthma, diabetic and fever. The medicinal valuable part of the Heen aratta plant is the rhizome.
The dry rhizomes are supplied to the Ayurveda medicine shops by local collectors and import from India like foreign countries. The collectors identify the Heen aratta plant by the external morphological characters. This method is not enough to identify the Heen aratta plant correctly, because there are many related species to Alpinia calcarata and their morphological characters are almost the same. The related species are Alpinia galanga (Maha aratta), Alpinia malaccensis (Ran kinihiriya) and etc.
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
3.1 Research location
3.2 Plant Materials
3.4 Equipment
3.5 Method
3.5.1 Dry Rhizome Samples Collection and Storage
3.5.2 DNA Extraction
3.5.2 DNA Quantification Using UV Spectrophotometer
3.5.3. Quantification Using Known DNA Sample
3.5.4 Restriction Digestion Reaction
3.5.5 PCR Amplification of Extracted Genomic DNA
CHAPTER 04
RESULTS AND DISCUSSIONS
4.1 DNA Extraction
4.2 DNA Quantification Using UV Spectrophotometer
4.3 Restriction Digestion Reaction
4.4 PCR Amplification
4.5 Estimated Cost Calculation
4.6 Time Consumption for DNA Extraction
CHAPTER 05
CONCLUSION
Research Objectives and Topics
This research aims to develop a cost-effective and efficient DNA extraction protocol for dry rhizomes of Alpinia calcarata (Heen aratta) to support accurate DNA barcoding and overcome issues related to secondary metabolites and high costs of commercial kits.
- Comparison of three different modified DNA extraction methods (SDS, 4x CTAB, and CTAB series).
- Quantification and purity assessment of extracted genomic DNA using UV spectrophotometry.
- Validation of DNA suitability for downstream applications through restriction digestion and PCR amplification.
- Evaluation of cost and time efficiency for each extraction protocol to identify the most suitable method.
Excerpt from the Book
Modified protocol by Porebski et al., 1997
A comparatively fast, cheap and coherent DNA extraction protocol comprising big amounts of polyphenols, tannins, and polysaccharides. The technique includes a modified extraction of CTAB, using elevated salt levels to remove polysaccharides, using polyvinyl pyrrolidone (PVP) to remove polyphenols, prolonged treatment with RNase, and extraction of phenol-chloroform. The existence of secondary metabolites like polyphenols, tannins, and polysaccharides inhibits the action of enzymes. Polysaccharides are visually obvious in DNA obtained by their vicious, glue-like texture and render DNA unmanageable in the polymerase chain response (PCR) by inhibiting Taq polymerase activity (Porebski et al., 1997).
The purity of DNA becomes increasingly hard when young, expanding leaf and shoot material is restricted or when the plant does not undergo active shoot elongation at the moment of collection. DNA from previous material was found to be hard to obtain and unstable for long-term storage. Leaves contain enhanced amounts of polyphenols, tannins, and polysaccharides with maturity. When younger growing leaves and shoots are not accessible during the collection moment, it becomes essential to deal with such parts in mature leaves. A DNA extraction protocol from fully developed extended leaf tissue that would produce DNA appropriate for PCR, particularly for RAPDs, and would be compatible for many distinct wild and grown strawberry species. The protocol outlined here is comparatively fast and cheap and offers smooth, continuously amplifiable DNA in the polymerase chain reaction using the RAPD method. Extraction buffer consist of, 100 mM tris, 1.4 M NaC1, 20 mM EDTA, pH 8.0, 2% CTAB, 0.3% ~-mercantoethanol (Porebski et al., 1997).
Summary of Chapters
CHAPTER 01 INTRODUCTION: This chapter outlines the medicinal importance of Alpinia calcarata and defines the research problem regarding the high cost and difficulty of extracting quality DNA from dry rhizomes due to secondary metabolites.
CHAPTER 02 LITERATURE REVIEW: Provides an overview of the Zingiberaceae family, plant identification, storage techniques for DNA extraction, and existing molecular methods for plants high in secondary metabolites.
CHAPTER 03 MATERIALS AND METHOD: Details the specific experimental procedures, equipment, and chemicals used to conduct the DNA extraction, quantification, and downstream validation.
CHAPTER 04 RESULTS AND DISCUSSIONS: Presents the findings regarding DNA concentration, purity, and cost-time efficiency of the tested extraction methods, including visual validation via gel electrophoresis.
CHAPTER 05 CONCLUSION: Summarizes that while all tested methods yielded suitable DNA, the SDS method was identified as the most cost-effective and time-efficient, surpassing the performance of expensive commercial kits.
Keywords
Cost-effective method, Heen aratta, DNA extraction, Restriction digestion, PCR amplification, Alpinia calcarata, Zingiberaceae, Secondary metabolites, Genomic DNA, UV spectrophotometry, Gel electrophoresis, CTAB, SDS.
Frequently Asked Questions
What is the primary focus of this research?
The research focuses on developing a low-cost, effective DNA extraction protocol for dry rhizomes of Alpinia calcarata (Heen aratta) for DNA barcoding purposes.
What are the central thematic areas covered?
The work covers molecular plant biology, specifically DNA isolation techniques, purification, quantification, and validation through downstream applications like PCR.
What is the main objective of the study?
The primary goal is to find a cost-effective alternative to expensive commercial DNA extraction kits that is specifically optimized for medicinal plants with high secondary metabolite content.
Which scientific methods were employed?
The study utilized modified SDS and CTAB-based extraction protocols, UV spectrophotometry for quantification, restriction enzyme digestion, and PCR amplification.
What topics are discussed in the main part of the thesis?
The main part covers the literature on Zingiberaceae, detailed extraction methodologies, cost estimation, time consumption analysis, and the verification of DNA quality through various laboratory assays.
Which keywords define this work?
Key terms include Cost-effective method, Heen aratta, DNA extraction, Restriction digestion, and PCR amplification.
Why are rhizomes difficult for DNA extraction?
Rhizomes typically contain high levels of phenolic compounds and secondary metabolites which can inhibit enzymes and cause the DNA to degrade or become unusable for downstream processing.
How was the cost-effectiveness determined?
The cost-effectiveness was determined by calculating the specific costs of chemicals required for each 20 ml of extraction buffer and comparing these against the price of commercial DNA extraction kits.
What was the outcome of the restriction digestion test?
The restriction digestion test, visualized by gel electrophoresis, showed clear smears for pure samples, confirming that the extracted DNA was of sufficient quality and free of significant inhibitors.
- Citation du texte
- Pasan Amarasinghe (Auteur), 2020, DNA Extraction from Dry Rhizomes for DNA Barcoding. Development of a Cost-Effective Method, Munich, GRIN Verlag, https://www.grin.com/document/1119261
