DNA Barcoding: a step by step guide

Summary

This video presents a simple, 12-step workflow for DNA barcoding to track biodiversity, using basic lab equipment. It covers the process from extracting DNA from a specimen, such as a bug's leg, to performing PCR and sequencing for species identification. Key steps include DNA extraction, quality checks, and sending samples for sequencing, using commercial kits and gel electrophoresis. The video highlights the importance of DNA barcoding in biodiversity research and showcases its potential for species discovery and understanding ecological dynamics, aided by advancements in technology and automation.

Takeaways

• 😀 Biodiversity is crucial for survival, but over 1 million species are threatened with extinction, leading to crises like new diseases and food production issues.
• 😀 Major research programs use molecular methods like DNA barcoding to track biodiversity on a global scale, measuring changes due to climate change and human activities.
• 😀 DNA barcoding can be done with basic lab infrastructure using commercial kits, particularly for DNA extraction.
• 😀 Step 1: Prepare all necessary equipment and reagents by carefully reading the chosen protocol before starting the process.
• 😀 Step 2: Collect a leg from the sample organism, label the tube, and change gloves to avoid contamination for the next steps.
• 😀 Step 3: Add lysis buffer and proteins K to break the cell membrane and inactivate proteins that may degrade DNA during the process.
• 😀 Step 4: Incubate the samples at the specified temperature and duration before a quick spin in the centrifuge to complete the tissue lysis.
• 😀 Step 5: Set up DNA extraction by adding binding mixture and using a membrane filter to isolate the DNA, followed by centrifugation and washing to purify the DNA.
• 😀 Step 9: Conduct a quality check of PCR products using gel electrophoresis to confirm successful amplification before sequencing.
• 😀 Step 12: Send the PCR products to a sequencing provider for Sanger sequencing, and compare the sequence with databases like BOLD to identify the species.
• 😀 Advances in DNA barcoding technologies, including automation and high-throughput sequencing, are revolutionizing species discovery and biodiversity research.

Q & A

What is the main purpose of DNA barcoding in biodiversity research?

DNA barcoding is used to identify and track species on a planetary scale, providing a baseline of biodiversity data that can help measure changes due to climate change and human activities.

Why is it important to change gloves between steps during the DNA barcoding process?

Changing gloves prevents cross-contamination of samples, which is critical for obtaining accurate DNA results.

What role does lysis buffer and Proteinase K play in DNA extraction?

Lysis buffer breaks down cell membranes, and Proteinase K inactivates proteins that could degrade DNA, ensuring that the DNA remains intact for extraction.

How is DNA purified after extraction from the sample?

DNA is purified by binding it to a membrane filter, washing away proteins and salts with wash buffer, air-drying the membrane, and eluting the DNA into collection tubes.

What is the purpose of including negative controls in PCR setup?

Negative controls for DNA extraction and PCR are included to detect any contamination that may have occurred during the setup, ensuring the reliability of results.

Why is gel electrophoresis used before sequencing the PCR products?

Gel electrophoresis is used to check the quality and success of the PCR amplification, allowing visualization of DNA bands to confirm that the target DNA was successfully amplified.

What is Sanger sequencing, and why is it used in this workflow?

Sanger sequencing is a highly accurate method for reading DNA sequences base by base. It is used to obtain precise DNA barcode sequences for species identification.

How are the sequenced DNA fragments used to identify species?

The DNA sequences are compared against databases like the Barcode of Life Data System (BOLD) to find matches and identify the species of the sample.

What advantages does high-throughput sequencing offer over the basic tube-based method?

High-throughput sequencing automates and scales up DNA barcoding, enabling rapid species discovery, exploration of species interactions, and tracking of biodiversity at an unprecedented speed.

What broader ecological or societal benefits does tracking biodiversity through DNA barcoding provide?

Tracking biodiversity helps detect threats such as invasive species, new diseases, and disruptions in food production, informs conservation strategies, and provides data to understand the impact of climate change and human activities on ecosystems.

Why is it recommended to use commercial kits for DNA extraction?

Commercial kits simplify the DNA extraction process, reduce the likelihood of errors, and ensure consistency and reliability in obtaining high-quality DNA.

What is the significance of the Barcode of Life Data System (BOLD) in DNA barcoding?

BOLD is a comprehensive repository for DNA barcode records, including specimen data, images, sequences, and trace files, allowing researchers to accurately identify species and share biodiversity data globally.