Single animal PCR genotyping protocol for C. elegans

Abstract: In model organisms, polymerase chain reaction (PCR) genotyping is essential for strain construction and verification. Typically, single animals are used for genotyping. While PCR is a routine laboratory technique, performing reproducible high-quality single animal PCR genotyping can be challenging, especially for researchers new to the technique. Here we describe a standard yet robust PCR procedure that can detect various types of genetic alleles using the genomic DNA template derived from single C.elegans animals. We included some considerations on handling reagents, to increase the chance of successful PCR and minimize background signal. We previously presented a PCR genotyping strategy to detect single nucleotide polymorphisms (Chen & Schedl, 2021). Here, we present a detailed PCR protocol for routine single animal genotyping in C. elegans.

Materials and Reagents:

1. Gotaq DNA polymerase and separate colorless and green PCR buffers (Promega, catalog number: M3008)

2. dNTPs (Promega, catalog number: U1515)

3. Single strand DNA oligoes (ordered from IDT)

4. Proteinase K (Gold Biotechnology, catalog number: P-480-1)

5. Agarose (Sigma, catalog number: A6013)

6. Tris (Sigma, catalog number: T6066)

7. Boric acid (Fisher Scientific, catalog number:BP168-1)

8. EDTA (Sigma, catalog number: ED2SS)

9. SYBR Safe DNA Gel Stain (Invitrogen, catalog number: S33102)

10.Milli-Q water, autoclaved or filter sterilized

11.PCR 8-well strips (Midwest, catalog number: PR-PCR28CF)

12.Veriti 96-Well Fast Thermal Cycler (Applied Biosystems, catalog number: 4375305)

13.Eppendorf tube (Genemate, catalog number: C-3260-1)

14.1 ml filtered plastic micropipette tips (USA Scientific, catalog number: 1122-1830)1

15.200 μl filtered plastic micropipette tips (Alkali Scientific, catalog number: FT1200)1

16. 20 μl filtered plastic micropipette tips (USA Scientific, catalog number: 1183-1810)1

17.1-10 μl filtered plastic micropipette tips (USA Scientific, catalog number: 1181-3710)1

Procedure:

A. Preparation of single animal genomic DNA template

1. Add 100 μl 5x colorless PCR buffer into 900 μl of Milli-Q water, invert to mix thoroughly².

2. Add 5 μl 20 mg/ml Proteinase K solution into 100 μl diluted PCR buffer prepared in A1, mix well.

3. Aliquot 5 μl of solution (e.g., lysis buffer) prepared in A2 to each PCR tube.

4. Place a single animal into each PCR tube with a worm-pick³.

5. Incubate PCR tubes at 65 ℃ for 45 minutes, then 95 ℃ for 15 minutes⁴. Hold at 4 ℃.

6. Centrifuge the PCR tube and then add 15 μl of Milli-Q water into each PCR tube⁵, mix well.

B. PCR on a thermocycler

1. Prepare the PCR master mix following the recipe in Table 1, mix well. Scale up as needed.

2. Aliquot 24 μl of solution prepared in B1 into each PCR tube.

3. Add 1 μl of genomic DNA template prepared in A6 into each PCR tube⁶.

4. Invert to mix well, then spin down the PCR mix.

5.  Start the thermocycler and let the samples cycle through: 95 ℃ for 2 minutes for initial denaturation, then

35  cycles of 95  ℃ for 20  seconds, 55  ℃ for 20 seconds, and  72  ℃ for 30 seconds⁷, followed  by  final extension at 72 ℃ for 2 minutes. Let the samples cool down to 4℃ before proceed to the next step.

6. The reaction mix can be resolved on agarose gel immediately or stored at -20 ℃ freezer for later use.

Table 1.                                                              

For 50 μl PCR master mix                    volume (μl) 

5x green reaction buffer                        10

2.5 mM dNTPs                                        2

10 μM forward_primer                            2

10 μM reverse_primer                             2

DNA polymerase                                    0.25
Milli-Q water                                           34

C. Gel electrophoresis:

1. Add 1 gram of agarose into 100 ml 1x TBE buffer, microwave to dissolve the agarose.

2. Let it cool at room temperate for 10 minutes before adding 5 μl of SYBR safe DNA dye.

3. Pour the gel and let the gel solidify for 15 minutes at room temperature.

4. Load 15 μl of samples in B6 to each well and run the gel at 100 Volts for 20 minutes⁸.

5. Acquire gel image with a UV apparatus.

Notes:

1. To minimize cross contamination, we use filtered micropipette tips for all steps in the protocol.
2. Two types of PCR buffers are included with the Gotaq DNA polymerase. Both can be used in regular PCR reactions. In this protocol, we primarily use the 5x colorless PCR buffer to prepare lysis buffer in step A1. The green color buffer was mainly used in PCR reactions, to eliminate the need of adding loading dye to PCR samples prior to gel electrophoresis. 

3. In this protocol, we are using genomic DNA template prepared typically from wild-type looking adult stage animals. Animals with mutant phenotypes (e.g., dumpy, sterile, etc.) or L4 stage animals can also be used. In all cases, check under the dissection microscope to make sure that the animal is in the lysis buffer. For  worm-picking and general tips on handling C. elegans, please refer to Stiernagle, 2006 for additional information.
4. The incubation at 95℃ inactivates the proteinase K.
5. Each genomic DNA template prepared in A6 is good for ~20 PCR reactions. Thus, a number of loci and alleles can be genotyped in each individual animal, depending on the primer set. The genomic DNA template can be used immediately or stored at -20℃freezers for later use. For a typical strain construction,  individual hermaphrodites are placed on plates and allowed to produce progeny (lay eggs for 12 – 36hrs), and then genotyped. Progeny animals from plates where the parental hermaphrodite had the correct genotype are then picked to propagate the strain or continue the strain construction.
6. We use multichannel pipette when genotyping larger numbers of genomic of DNA samples. For each set of PCR, we always include necessary control samples to facilitate the interpretation of the PCR result. Examples of controls include: 1) positive control that contains a target allele known to work previously, to ensure PCR reagents are working. 2) negative control that doesn’t contain the experimental target allele, to determine the binding specificity of primers used in the PCR reactions. 3) control that contains no genomic DNA template, to ensure that solutions used and pipettes are not contaminated with DNA of unknown source.
7. We use ApE- A plasmid Editor software to design primers. For all primers used in the protocol, we choose primer length from nucleotides to 27 nucleotides. We choose the primers that have melting temperature around 60℃. The DNA polymerase amplify DNA at a rate of 1 kilobase per minute, adjust the time of the extension step based on your amplicon size. Conditions described are for amplicons have size range from 200 base pairs to 600 base pairs. We have not found it necessary to perform nested PCR with this protocol.
8. Check the gel to determine if longer period of electrophoresis is required, depending on the type of allele genotyped.

Buffer recipes:

20 mg/ml Proteinase K

Add 1 gram of Proteinase K into 50 ml of Milli-Q water; mix well, aliquot, and stored at -20℃ freezer.
 

2.5 mM dNTPs

Add 1 ml of 10 mM dNTPs into 4 ml of autoclaved water; mix well,aliquot into smallsingle use volume,

stored at -20℃ freezers. Dispose and thaw a new tube(s) for each experiment.

10xTBE buffer

Dissolve 108 grams of Tris, 55 grams of Boric acid, and 9.3 grams of EDTA in water 109 to make a final 1 liter of the 10xTBE buffer.

References:
Chen, J., & Schedl, T. (2021). A simple one-step PCR assay for SNP detection. MicroPublication Biology, 2021. https://doi.org/10.17912/MICROPUB.BIOLOGY.000399
Stiernagle, T. (2006) Maintenance of C. elegans, WormBook, doi/10.1895/wormbook.1.101.1