DNA barcode collection-analysis protocol experiment

Specimen and tissue processing
Barcode analysis of most specimens is straightforward but highly dependent on the initial conditions of the D NA. Therefore, care must be taken to ensure that specimens are executed in a manner that does not damage DNA, that they are analyzed as soon as possible after collection (see Note 1), and that precautions are taken to prevent contamination with other types of DNA (see Note 2). There are six key steps to ensure that each specimen is handled and archived correctly.

1. Organize specimens into lots of 94 (see Note 3). Laser print a small label with an individual registration number for each of the 9 4 specimens. If a voucher specimen is to be preserved, attach a label with its registration number to it or to its container.

2. Clean the work surface with ethanol or detergent to remove DNA and DNAzyme contamination.

3. For each specimen, remove a small piece of tissue using acid or flame-sterilized forceps and/or a scalpel. Place the sample in a separate preservation tube in the TrakMates box and label the specimen with the appropriate specimen registration label.

4. For room temperature storage, fill the tube with ethanol. For dry tissues (e.g., insect legs) or tissues to be stored frozen, additional ethanol may not be necessary.

5. record the specimen registration number and related data on a spreadsheet program (for the spreadsheet program and filing, see w w w .barcodeoflife.org).

6. photograph each specimen.

Genomic DNA Extraction/Purification

There are many alternative methods for genomic DNA extraction and/or purification (see Note 4). Many of these methods have been thoroughly tested for their efficiency in high-throughput animal DNA barcoding (2 4 ); two very efficient methods for fresh/frozen or archived tissues are described below.

5. Add sequencing reactants (~10 u L ) to the center of the dextran gel in each well using a multichannel pipette.

6. Using a multichannel pipette and an 8-well PCR tube strip, add the sequencing reagent (~10 u L ) to each well of a sterile 96-well reaction plate.

1 0 / xL formamide to each well of a sterile 96-well reaction plate.

7. Elute the purified sequencing reaction into formamide, place the reaction plate under the dextran gel filter plate, secure with a rubber band, and centrifuge at 750 g for 3 m i n .

3.7 Sequencing Analysis

D N A barcoding devices require high-throughput sequencing. Applied Biosystem and A m e r s h a m have several reliable devices with different sequencing capabilities. The Applied Biosystem 3730 Capillary Sequencer was used in the following program.

1. Cover the reaction plate with an adhesive pad.

2. Put the reaction plate on the base and install the positioner.

3. On the assembled plate, print and paste a bar code.

4. Place the assembled board in the 3730 instrument.

5. Perform routine 3730 servicing as needed.

6. Use the board manager of the data collection system to enter the board number.

7. Start the run in the Run Scheduler.

3.8 Sequencing editing/comparison

The basic functions of sequence editing and comparison software packages for analyzing high-throughput barcodes include collecting bidirectional data and editing trace files. Software such as Sequencher, SeqScape, and Lasergene have this functionality, as well as other features.

1. Using the program or software of choice, open the trace file after sequencing is complete. If the chosen software is powerful, the samples can be analyzed in a single run.

2. Collect forward and reverse reads for each sample.

3. remove primer sequences from each set of data.

4. Carefully proofread each base name and make appropriate adjustments for misnamed or unnamed bases.

5. Save the sequence in Fasta format and upload it to a suitable project or other online sequence library.

1. Treatments that do not damage DNA are freezing, cyanide treatment or ethanol immersion; try to avoid treatments with ethyl acetate or formalin, which can damage DNA. DNA can usually be stored in dehydrated samples for at least one year, after which it will slowly degrade. DNA in frozen samples (especially when stored at low temperatures) remains stable for long periods of time. However, DNA in ethanol-preserved samples is often degraded by acidification. Therefore, barcode analysis is best performed immediately after sample collection.

2. Samples should be handled on a clean surface, and the tissue processing equipment used should be acid or flame sterilized after processing each sample. Two blank wells should be set aside for positive and negative controls, and the remaining 94 wells should be used for samples, taking care to avoid cross-contamination between wells when adding samples.

3 . In any laboratory where high yields are desired, all barcode analysis steps must be performed in a 96-well plate. Procedure

4. In addition to the chelex- and membrane-based methods discussed here, there are many other kits available for D N A isolation and release. The use of magnetic beads is becoming more common, primarily because of the ease of automating their operation. In time-critical situations, there are several kits that guarantee D N A extraction within a few minutes, such as the Extract-N - A m p P C R Kit (Sigma-Aldrich) and F T A cards (Waterman-Florham Park, N J ). These methods are now being evaluated for feasibility for high-throughput D N A barcoding methods.

5. D r y R ekase is a chelex-based method for isolating D N A that rapidly releases D N A in solution for downstream applications (24). It requires minimal tissue samples and shortens processing time, but is not suitable for samples containing high levels of PCR inhibitors (e.g., hemoglobin) and for samples in which DNA has been degraded, as well as for purified DNA that needs to be preserved for long periods of time.

6. The reaction volume depends on the sample volume and can range from 30 to 100 ^L. For example, organ tissues such as legs of small crustaceans or small insects require only 30 juL of extract. For example, organ tissues such as legs of small crustaceans or small insects require only 30 juL of extraction solution, while small pieces of vertebrate tissues can be lysed with 100~110 uL of extraction solution.

7. The Nucleospin 96 Tissue Kit is a membrane-based DNA extraction method that depends on the affinity of DNA for silica membranes at high salt concentrations. This method is very sensitive and yields high purity DNA and can be used to study degraded DNA samples. The high cost and time required limit its use.

8. With this method, as with most other kits, a manual or motorized multichannel pipette is required for efficient extraction of DNA from 96-well plates. Similarly, almost all kits and procedures for 96-well plates can be performed using an automated dispenser workstation.

9. When using the DryRelease method, a small amount of distilled water is sufficient to elute a small number of specimens (or samples in which DNA is likely to be degraded), whereas a large amount of water is required when using fresh or bulky samples.

10. Centrifuge % well plates on square centrifuge modules or in open MN test tube racks to avoid breakage by centrifugal force.

11. Primer design is critical and small changes can have a dramatic effect on barcode synthesis. The first step in studying a new population is to identify an optimized primer. To resolve mismatches between primers and template DNA, it is recommended to use simple primers or hypoxanthine-containing primers (26). By utilizing primers containing 2 to 4 simple or hypoxanthine bases, better barcode amplification can be obtained from complex templates and pseudogene amplification in the nucleus can be prevented (27).

12. The use of sterilized pipette tips with stoppers is recommended for all PCR reagent-related operations to avoid unnecessary contamination. Clean the benchtop with alcohol or detergent before starting experimental procedures. Keep DNA templates (DNA extracts or PCR products) away from PCR reagents until the reaction has been set up. Add the DNA template after all reagents have been removed and returned to the refrigerator.

13. The addition of alginate is optional, but it may increase the chance of a successful PCR reaction (28) (Fig. 4) and may facilitate freezing of the dispensed master mix. The mixture can be stored at -20- C for ~3 months.

or dispensed directly into 96-well plates and stored at 20°C for 1 month.

14. The reaction system can be significantly reduced to minimize costs. To accurately dispense minute quantities of reagents, prepare a sufficient number of

master mix for several plates is necessary. Note that additional reaction solutions must be available to make up for the excess of reagents.

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