Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Summary

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Abstract

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Introduction

Here, two related protocols are presented for the lysis of Caenorhabditis elegans to make DNA accessible for PCR-based applications. PCR is a commonly used molecular technique used for many applications, including genotyping and amplifying DNA fragments for cloning and sequencing, among others. The small (1 mm), free-living roundworm C. elegans is a popular animal system for biological research. Obtaining suitable genomic DNA from a single animal or a few animals is sufficient to amplify the sequence by PCR. Late L4 larvae and adults contain only ~1,000 somatic cells (including some multi-nuclear, polyploid cells), germ cells, and (if the animal is a gravid hermaphrodite) offspring in utero¹. However, these animals are protected by a cuticle that must be disrupted to extract the genomic DNA². Standard methods to prepare nematode genomic DNA template for PCR involve multiple steps and take several hours. The animals are first frozen in worm lysis buffer containing proteinase K (−70 °C or below) for at least 15-45 min (longer is recommended by some protocols)^3,4,5,6. This step cracks open the animals.

After freezing, the animals are incubated for 1 h at 60-65 °C for the proteinase K to work, then the enzyme is inactivated for 15-30 min at 95 °C. The proteinase K destroys the nucleases that degrade DNA. The inactivation of proteinase K before PCR is important to prevent the proteinase K from degrading the DNA polymerase. The two kit-based protocols described here are quick, reliable, and cost-effective methods to extract genomic DNA from either a single animal or a few nematodes for everyday research and teaching laboratory applications. The kit used was originally optimized by the manufacturer to extract DNA from animal tissue, saliva, and hair⁷. It uses a proprietary tissue preparation solution and extraction solution to lyse cells and make genomic DNA accessible. A proprietary neutralization solution then neutralizes the components that may inhibit PCR (e.g., salts, ions, and Mg²⁺-binding molecules).

When genotyping, a single animal can be tested. When determining if a strain is homozygous, testing six or more offspring from a single animal gives high confidence that a line is homozygous or not (there is a 0.02% chance of randomly picking six homozygous mutant progeny from a heterozygous parent [(1/4)⁶ × 100% = 0.02%]). This method 1) is straightforward, with fewer steps than the proteinase K method, and 2) decreases the template preparation time to 15 min. The results in this work demonstrate that the developed protocol works robustly in extracting genomic DNA from single or a few worms, which can be reliably used for downstream applications that do not require highly purified DNA, including PCR.

Protocol

1. C. elegans maintenance

NOTE: N2 (wild type) and blmp-1(tm548) C. elegans strains were maintained on standard nematode growth media (NGM) plates at 20 °C.

1. Prepare NGM plates by dissolving 23.005 g of NGM powder in 973 mL of water in a 2 L flask. Cover the flask opening with aluminum foil (or autoclavable cap that allows venting) and secure the covering with autoclave tape. Autoclave to dissolve the powder and sterilize the contents with a sterilization cycle of at least 30 min.
2. Cool the medium to 60 °C in a water bath.
3. To the cooled medium, add 25 mL of sterile 1 M phosphate (KH₂PO₄) buffer, pH 6.0; 1 mL of 1 M calcium chloride solution; and 1 mL of 1 M magnesium sulfate solution.
4. Stir the medium on a magnetic stir plate to obtain a homogeneous mixture and to prevent uneven cooling and solidification (~5 min). Ensure that the stir bar spins fast enough to create a vortex but not so fast that bubbles are introduced (~250-300 rpm).
5. Pour ~25 mL of the medium into each 10 cm Petri plate (~40 plates in total). Dry the plates at room temperature overnight (typically 16-25 °C).
6. Grow a colony of Escherichia coli OP50 in 30-50 mL of LB broth overnight at 37 °C.
7. Seed each plate with 500 µL of E. coli OP50 culture and let them dry at room temperature before use (typically 16-25 °C)⁸. Store the plates at 4 °C for up to 3 weeks.
8. Transfer C. elegans to seeded plates using a wire pick or other method and let them grow to L4 or adult stage at the temperature required for the strain (typically 16-25 °C, 1-2 days if animals were plated starved as dauers, 3-4 days if animals were plated as embryos)⁸.

2. Single-worm DNA extraction

NOTE: This method is useful for extracting DNA from a single worm (one worm in 1.8 µL of total volume). A master mix can be made if multiple worms will be lysed at one time.

1. Program a thermocycler for one cycle at 55 °C for 10 min followed by 95 °C for 3 min (lysis program).
2. Aliquot 0.8 µL of the extraction solution from the kit onto the inside wall of a 0.2 mL PCR tube on ice. Add 0.2 µL of the tissue preparation solution from the kit to the droplet of the extraction solution. Mix by pipetting.
3. Identify an L4 or adult animal under a dissecting microscope⁹. To identify L4 hermaphrodites, look for a characteristic vulva that is visible as a pale half-circle in the middle of the animal. Identify adult hermaphrodites by looking for the largest animals on the plate that may have oval embryos visible in their uterus.
4. Using a platinum wire pick, transfer the selected animal into the solution¹⁰.
5. Centrifuge the tube briefly (2-3 s, ≤6,000 rpm/2,000 × g) at room temperature to collect the contents at the bottom of the tube.
6. Place the tube in the thermocycler and run the lysis program set at Step 2.1.
7. When the program is complete, briefly centrifuge the tube and place it on ice. Add 0.8 µL of the neutralization solution from the kit to the mixture. Mix by pipetting.
8. Centrifuge the tube briefly at room temperature (2-3 s, ≤6,000 rpm/2,000 × g).
9. Directly use the lysate for PCR or store it at 4 °C or −20 °C for future use.
   ​NOTE: Extracted DNA is stable at 4 °C or −20 °C for at least 6 months⁷.

3. DNA extraction of a few individuals

NOTE: This method is useful for extracting DNA from a few worms. A master mix can be prepared if multiple strains will be lysed at one time.

1. Program the thermocycler for one cycle at 55 °C for 10 min followed by 95 °C for 3 min (lysis program).
2. Aliquot 2.0 µL of the extraction solution from the kit onto the inside wall of a 0.2 mL PCR tube on ice. Add 0.5 µL of the tissue preparation solution from the kit to the droplet of extraction solution. Mix by pipetting.
3. Identify L4 or adult animals under a dissecting microscope⁹. L4 hermaphrodites have a characteristic vulva that is visible as a pale half-circle in the middle of the animal. Adult hermaphrodites are the largest animals on the plate and may have oval embryos visible in their uterus.
4. Using a platinum wire pick, transfer a few animals into the solution: 9 animals yield 1 animal equivalent of genomic DNA per 0.5 µL of lysate, and 16 animals yield ~1 animal equivalent of genomic DNA in 0.25 µL (3.5 nematodes/µL)¹⁰.
   NOTE: It is difficult to transfer greater than 16 animals into this volume.
5. Centrifuge the tube briefly at room temperature (2-3 s, ≤6,000 rpm/2,000 × g).
6. Place the tube in the thermocycler and run the lysis program set at Step 3.1.
7. When the program is complete, briefly centrifuge the tube and place it on ice. Add 2 µL of the neutralization solution from the kit to the mixture. Mix by pipetting.
8. Centrifuge the tube briefly at room temperature (2-3 s, ≤6,000 rpm/2,000 × g).
9. Directly use the lysis for PCR or store it at 4 °C or −20 °C for future use.
   ​NOTE: Extracted DNA is stable at 4 °C or −20 °C for at least 6 months⁷.

4. PCR reaction

NOTE: One downstream application of this worm lysis technique, detecting a deletion mutation using a fast polymerase, is described. The effectiveness of the two worm lysis protocols in producing genomic template DNA for successful PCR at dilutions to 1/50^th of a worm per reaction is also demonstrated.

1. Extract DNA from a single worm and 16 worms using wild-type N2 and mutant strains, as described above in Step 2 and Step 3.
2. Prepare 2-, 10-, 20-, and 50-fold dilutions of single-worm DNA from wild-type N2 animals.
3. Set up PCR reactions following the manufacturer's protocol using primers specific to the sequence of interest and hot-start PCR master mix (Table 1 and Table 2). Use 1 µL of undiluted DNA or 2 µL of diluted DNA as the template in each reaction.
4. Confirm the PCR products by gel electrophoresis and imaging¹¹.

Results

Genomic DNA from a single or a few wild-type adults was extracted using the commercial kit or traditional lysis protocol to compare the efficacy of these two methods. These lysates were then used as templates for PCR to amplify either a larger target of ~2,100 bp (encoding blmp-1) or a smaller target of ~500 bp (encoding a part of sma-10). Both methods successfully yielded appropriate PCR products (Figure 1A).

Next, the ability of kit-extracted g...

Discussion

Determining the genotypes of C. elegans is an important step while performing genetic crosses to create new C. elegans strains. Genomic DNA extraction using a single or few C. elegans is a crucial step in genotyping C. elegans. This protocol describes genomic DNA extraction from C. elegans using a commercial kit. This method is fast and works robustly. The genomic DNA extracted using this method can be used for downstream applications, including genotyping, sequencing, and clo...

Materials

Name	Company	Catalog Number	Comments
autoclave tape	Defend	43237-2
aluminium foil, heavy duty	Reynolds Wrap	2182934
calcium chloride	Millipore Sigma	102382 (CAS 10035-04-8)
Extract-N-Amp kit (includes Tissue Preparation Solution, Extraction Solution, and Neutralization Solution)	Sigma-Aldrich Co. LLC	XNAT2-1KT
Isotemp hotplate/stirrer	Fisher Scientific	11-100-495H
LB media, Lennox, capsules	MP Biomedicals, LLC	3002-131
magnesium sulfate, 97% pure, anhydrous	Thermo Scientific	AC413485000 (CAS 7487-88-9)
microcentrifuge	Labnet International, Inc.	PrismR, C2500-R
NEB Q5U Hot Start High-Fidelity DNA polymerase	New England Biolabs, Inc.	M0515S	"Pol E" used in Supplemental Figure S1, a high-speed, high-fidelity polymerase (20–30 s/kb)
NGM media powder	US Biological Life Sciences	N1000
Phusion High-Fidelity PCR Master Mix with HF Buffer	New England Biolabs, Inc.	M0531S	"Pol D" in Figure 1B, a high-speed, high-fidelity polymerase (15–30 s/kb)
primers	Integrated DNA Technologies	custom DNA oligos
PrimeSTAR GXL polymerase	Takara Bio Inc.	R050B	"Pol C" in Figure 1B, a high-fidelity polymerase (1 min/kb) for GC-rich templates and templates up to 30 kb
Quick-Load Purple 2-log DNA Ladder (0.1–10.0 kb)	New England Biolabs, Inc.	N0550S
SapphireAmp Fast PCR Master Mix	Takara Bio Inc.	RR350A	"Pol A" in Figure 1B, polymerase used in Figure 1A, C, D, a high-speed polymerase (10 s/kb) for targets up to 5 kb
Sigma ReadyMix Taq PCR reaction mix	Sigma-Aldrich Co. LLC	P4600	"Pol B" in Figure 1B, a polymerase (1 min/kb) for targets up to 7 kb
SimpliAmp thermal cycler	Applied Biosystems	A24812
stir bar	Fisher Scientific	14-512-126
vortex mixer	Fisher Scientific	2215365
worm pick	Genesee Scientific Corporation	59-AWP