Single Worm PCR: A Method to Extract and Amplify Genomic DNA

Overview

This video describes a technique to rapidly screen a single nematode for a genetic marker by PCR screening. In the example protocol, we screen for CRISPR-based genome editing.

Protocol

The following protocol is an excerpt from Prior et al, A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins, J. Vis. Exp. (2018).

Single Worm PCR and Genotyping

1. After 1 - 2 days of egg-laying, transfer the F₁s into individual PCR strip tube caps containing 7 µL of Worm Lysis Buffer using a worm pick (Table 1, Figure 1D, day 4 - 5).
2. Centrifuge PCR tubes at maximum speed for 1 min at room temperature to bring animals to the tube bottom, and freeze tubes at -80 °C for 1 h.
   NOTE: Worms can be stored at -80 °C indefinitely.
3. Lyse frozen worms in a thermocycler using the following program: 60 °C for 60 min, 95 °C for 15 min, 4 °C hold.
4. Set-up PCR mastermix as shown in Table 2.
5. Add 21 µL of PCR mastermix into clean PCR tubes, and then add 4 µL of worm lysis from step 3. Mix well using a pipette and run PCR program following the manufacturers guidelines (see Materials Table).
6. Purify PCR reactions using a DNA Clean and Concentrate kit, following the manufacturer instructions (see Materials Table). Elute DNA by adding 10 µL water to the spin column.
7. Set-up restriction enzyme mastermix as shown in Table 3.
8. Add 10 µL of the restriction enzyme mastermix to each cleaned PCR reaction and incubate for 1 - 2 h at 37 °C.
9. Separate digested PCR products on a 1.5% agarose gel run at 120 V using 1x Tris-acetate-EDTA (TAE) buffer.

Representative Results

Figure 1. CRISPR-Cas9 engineering of the C. eleganssod-1 locus. (A) Schematic illustration depicting the exon-intron structure of the sod-1 locus in C. elegans. The red bar denotes the location of G93 in exon 3. The primer pair set is noted by the red arrows (F1-R1). (B) Sequence alignment of human and C. elegans (worm) SOD-1 proteins. The targeted G93 residue is highlighted in red. (C) Top, a section of genomic DNA surrounding the G93 codon is shown as a reference, and the PAM (green) and sgRNA target (blue) sites are highlighted. Bottom, the single stranded oligonucleotide (ssODN) homology directed repair (HDR) template is shown containing: the codon edit changing G93 to A93, a silent change to the PAM motif to prevent cleavage by the sgRNA-Cas9 complex, a silent change to introduce a unique HindIII restriction enzyme site (yellow box), and flanking 5' and 3' 50 bp homology arms (black bars). All nucleotide changes designed in the ssODN are highlighted red. (D) Schematic illustration of the pipeline for generating and identifying CRISPR-Cas9 RNP-based point mutants. On day 0, inject 10 - 15 P₀ animals with injection mix. On day 2 - 3, identify successfully injected P₀ animals by those containing fluorescent F₁ progeny, and select three P₀ plates with the most fluorescent progeny. From each of the three P₀ plates, single 8 fluorescent F₁ progeny. On day 4 - 5, (a) step 1, individual F₁s are picked and placed into PCR tubes containing Lysis Buffer; (b) step 2, after freezing at -80 °C, PCR tubes containing F₁ worms are lysed to release genomic DNA, which is used as a PCR template. The PCR products are then cleaned and digested with the unique restriction enzyme; (c) step 3, enzyme digested products are resolved on an agarose gel where wild type (+/+), heterozygous (m/+), and homozygous (m/m) animals can be identified by restriction digest banding patterns. Please click here to view a larger version of this figure.

Reagent	[Final]
KCl	50 mM
Tris-HCl pH 8.3	10 mM
MgCl2	2.5 mM
NP-40	0.45%
Tween-20	0.45%
Proteinase K	1 mg/mL

Table 1. Worm Lysis Buffer Recipe. Proteinase K should be added fresh before each use.

Reagent	1x	24x
2X Q5 Mastermix	12.5 μL	300 μL
Primer F1 (10 μM)	1.25 μL	30 μL
Primer R1 (10 μM)	1.25 μL	30 μL
Worm Lysis	4 μL	----
ddH2O	6 μL	144 μL
Final Volume	25 μL	504 μL

Table 2. PCR Mastermix. The PCR mastermix should be mixed well by pipetting until the solution is homogeneous. 21 µL of the PCR mastermix should be added to clean PCR tubes, and then 4 µL of individual worm lysate should be added to properly labeled tubes.

Reagent	1x	24x
10X Enzyme Buffer	2 μL	48 μL
Cleaned PCR Reaction	10 μL	----
ddH2O	7 μL	168 μL
Restriction Enzyme (10 U/μL)	1 μL	24 μL
Final Volume	20 μL	240 μL

Table 3. Restriction Enzyme Mastermix. The restriction enzyme mastermix should be mixed well by pipetting until the solution is homogeneous. 10 µL of the restriction enzyme mastermix should be added to 10 µL of cleaned PCR product.

Materials

Name	Company	Catalog Number	Comments
Nuclease-free water	Synthego Inc.	provided with the sgRNA kit EZ kit
KCl	Sigma	P5405
DNA Clean & Concentrator	Zymo Research	D4004
Zymoclean Gel DNA Recovery Kit	Zymo Research	D4002
Q5 Hot Start High-Fidelity 2X Master Mix	New England Biolabs	M0494L
Proteinase K	Sigma	P2308
MgCl2	Sigma	M2393
NP-40	Sigma	74385
Tween-20	Fisher Scientific	BP337-100
RNaseZap Decontamination Solution	Fisher Scientific	AM9780