# Identification of Insertion Site by RESDA-PCR in Chlamydomonas Mutants Generated by AphVIII Random Insertional Mutagenesis

Fantao Kong, Yonghua Li-Beisson

► To cite this version:

Fantao Kong, Yonghua Li-Beisson. Identification of Insertion Site by RESDA-PCR in Chlamydomonas Mutants Generated by AphVIII Random Insertional Mutagenesis. Bio-protocol, 2018, 8(3), 10.21769/BioProtoc.2718. hal-03429789

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<figure><img src="image_1.png" /></figure>
# Identification of Insertion Site by RESDA-PCR in *Chlamydomonas*
## Mutants Generated by AphVIII Random Insertional Mutagenesis

Fantao Kong¹, ², ³, * and Yonghua Li-Beisson¹, ², ³

¹Commissariat à l'Energie Atomique et aux Energies Alternatives, Institut de Biologie Environnementale et Biotechnologie, CEA Cadarache, Saint-Paul-lez-Durance, France; ²Centre National de la Recherche Scientifique, UMR7265, Saint-Paul-lez-Durance, France; ³Aix-Marseille Université, UMR7265, Marseille, France

*For correspondence: [kongfantaohit@163.com](mailto:kongfantaohit@163.com)

**[Abstract]** *Chlamydomonas reinhardtii* is frequently used as a model organism to study fundamental processes in photosynthesis, metabolism, and flagellar biology. Versatile tool boxes have been developed for this alga (Fuhrmann *et al.*, 1999; Schroda *et al.*, 2000; Schroda, 2006). Among them, forward genetic approach has been intensively used, mostly because of the high efficiency in the generation of hundreds of thousands of mutants by random insertional mutagenesis and the haploid nature therefore phenotypic analysis can be done in the first generation (Cagnon *et al.*, 2013; Tunçay *et al.*, 2013). A major bottleneck in the application of high throughput methods in a forward genetic approach is the identification of the genetic lesion(s) responsible for the observed phenotype. In this protocol, we describe in detail an improved version of the restriction enzyme site-directed amplification PCR (RESDA-PCR) originally reported in (González-Ballester *et al.*, 2005). The improvement includes optimization of primer combination, the choice of DNA polymerase, optimization of PCR cycle parameters, and application of direct sequencing of the PCR products. These modifications make it easier to get specific PCR products as well as speeding up subcloning steps to obtain sequencing data faster.

**Keywords:** *Chlamydomonas reinhardtii*, Insertional mutagenesis, RESDA PCR, Forward genetic, *Chlamydomonas* mutant

**[Background]** In addition to the restriction enzyme site-directed amplification PCR (RESDA-PCR) (González-Ballester *et al.*, 2005), several other molecular techniques have been developed to identify insertion sites within the nuclear genome, including Genome Walker (Stirnberg and Happe, 2004), thermal asymmetric interlaced PCR (TAIL-PCR) (Dent *et al.*, 2005), 3'-rapid amplification of cDNA ends (3'RACE) (Meslet-Cladiere and Vallon, 2012), Mme1-based insertion site sequencing strategy (ChlaMmeSeq) (Zhang *et al.*, 2014), or whole-genome resequencing (Goold *et al.*, 2016). RESDA-PCR is based on the use of specific primers of the marker gene combined with the use of degenerate primers that anneal with sequences of restriction sites highly and randomly distributed in the nuclear genome. RESDA-PCR is one of the most commonly used, is not too expensive and has been found to give the highest possibility in identifying the flanking sequence in our hands.
Materials and Reagents
=======================

1. Falcon conical centrifuge tubes, 15 ml (Corning, catalog number: 430055)

2. Eppendorf tube, 1.5 ml and 2.0 ml, Eppendorf Quality™ (Eppendorf, catalog numbers: 0030120086 and 0030120094)

3. Petri dishes, 90 mm in diameter (Thermo Fisher Scientific, Sterilin™, catalog number: 101R20)

4. Sterilized toothpick (Fujian Fuhua, FUHUA FANGYUAN™, catalog number: 855)

5. *Chlamydomonas reinhardtii* mutants generated by random insertional mutagenesis (Cagnon *et al.*, 2013)

6. One Shot™ TOP10 Chemically Competent cell (Thermo Fisher Scientific, catalog number: C404010)

7. Zero Blunt™ TOPO™ PCR Cloning Kit (Thermo Fisher Scientific, catalog number: 450245)

8. Taq DNA polymerase (5,000 U ml⁻¹), 10x Standard Taq Reaction Buffer, dNTPs (New England Biolabs, catalog number: M0273S)

9. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301)

10. PCR-grade H₂O (Sigma-Aldrich, catalog number: W1754)

11. Ethanol (Sigma-Aldrich, catalog number: 34852)

12. KOD Xtreme Hot Start DNA polymerase, dNTPs (2 mM each), 2x Xtreme buffer (Merck, Novagen®, catalog number: 71842)

13. Agarose (Sigma-Aldrich, catalog number: A9539)

14. ExactLadder® DNA PreMix 2 log (OZYME, catalog number: OZYC002-500)

15. SYBR™ Safe DNA gel stain (Thermo Fisher Scientific, catalog number: S33102)

16. UltraPure™ 10x TAE buffer (Thermo Fisher Scientific, catalog number: 15558026)

17. Luria broth (Sigma-Aldrich, catalog number: L1900)

18. Kanamycin sulfate (Thermo Fisher Scientific, catalog number: 11815024)

19. NucleoSpin® Gel and PCR Clean-up Kit (MACHEREY-NAGEL, catalog number: 740609.10)

20. NucleoSpin® Plasmid Miniprep Kit (MACHEREY-NAGEL, catalog number: 740588.10)

21. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771)

22. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888)

23. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541)

24. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884)

25. Tris base (Sigma-Aldrich, catalog number: T6066)

26. Hexadecyltrimethyl ammonium bromide (CTAB) (Sigma-Aldrich, catalog number: H9151)

27. Isopropanol (Sigma-Aldrich, catalog number: W292907)

28. Phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma-Aldrich, catalog number: P3803)

29. Proteinase K (20.0 mg ml⁻¹) (Sigma-Aldrich, catalog number: P6556)

30. Lysis buffer (see Recipes)

a. 0.7 M NaCl

b. 10% SDS
c. 0.5 M Tris-HCl, pH 8.0

d. 10 mM EDTA

31. 5 M KCl (see Recipes)

32. 10% CTAB (see Recipes)

33. 10% DMSO (see Recipes)

34. 1.0% agarose gel (see Recipes)

35. 1x TAE (see Recipes)

36. 70% ethanol (see Recipes)

## Equipment

1. Shaker (Eppendorf, New Brunswick™, model: Innova® 44, catalog number: M1282-0002)

2. Accumet™ pH meter (Fisher Scientific, model: 3-in-1 Set, catalog number: 13-636-AE153)

3. PCR Thermal Cyclers (Thermo Fisher Scientific, Applied Biosystems™, model: 2720, catalog number: ED000651)

4. Agarose electrophoresis tank (Bio-Rad Laboratories, model: Mini-Sub® Cell GT, catalog number: 1704401)

5. Gel Doc XR System (Bio-Rad Laboratories, model: Gel Doc™ XR+, catalog number: 5838)

6. Fisherbrand™ Common bench-top vortexer (Fisher Scientific, catalog number: 02-216-125)

7. Bench-top centrifuge (Beckman Coulter, model: Allegra® 64R, catalog number: 367586)

8. Bench-top incubator (Eppendorf, New Brunswick™, model: S41i, catalog number: S41I230011)

9. NanoDrop 2000 (Thermo Fisher Scientific, model: NanoDrop™ 2000, catalog number: ND-2000)

10. Multisizer 3 Coulter counter (Beckman Coulter, model: Multisizer™ 3, catalog number: 6605697)

## Software

1. SPSS program (version 19.0)

## Procedure

### A. DNA extraction and quantification

1. Cultivate cells of *Chlamydomonas reinhardtii* (around $0.5 \times 10^6$ cells ml$^{-1}$) in shake flasks at 25 °C under constant white fluorescent light (100 µmol photons m$^{-2}$ m$^{-1}$) in TAP liquid media (Harris, 2001) with gentle shaking. Normally it will reach exponential phase (around $5 \times 10^6$ cells ml$^{-1}$) after 24 h.

2. Isolate and purify the genomic DNA of the mutant with the CTAB method (Schroda *et al.*, 2001). Briefly, after cells grow to logarithmic phase (around $5 \times 10^6$ cells ml$^{-1}$), harvest a total of $5 \times 10^7$ cells by centrifugation and suspend in 500 µl lysis buffer (100 mM Tris-HCl, pH 8.0, 1.75 mM EDTA, 150 mM NaCl, 2% [w/v] SDS, and 1.0 mg ml$^{-1}$ Proteinase K).
3. Incubate the lysates for 2 h at 55 °C, and then add 80 µl of 5 M KCl and 70 µl of preheated 10% CTAB and incubate for 10 min at 65 °C.

4. Extract the lysates twice with (500 µl) phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v), and then add (500 µl) chloroform to eliminate the remaining phenol.

5. The nucleic acids are precipitated for 20 min on ice by addition of 500 µl of isopropanol, and washed with 70% cold ethanol.

6. Resuspend the air-dried pellets in 50 µl sterile water.

7. Quantify the purified DNA using NanoDrop 2000, and add water to adjust the final concentration to be of 50 ng µl⁻¹.

*Note: The concentration of the cells mentioned is measured with Multisizer 3 Coulter counter.*

## B. PCR amplification

This procedure largely follows the protocol first published by González-Ballester *et al.* (2005). To identify the DNA sequence around the site of insertion, two subsequent sets of PCR reactions are required, named here as the 1st and 2nd amplification. The 1st PCR amplification is to amplify a fragment from genomic DNA of the mutant using one *AphVIII* gene-specific primer with one of the degenerate primers (Figure 1). To increase the chances of amplification, 4 degenerate primers can be tested, and each of them contains a sequence-specific part (Q0 at the 5' end) and a degenerate part (3' end). The PCR product will be used as a template for a second round of PCR amplification (the 2nd PCR amplification) with the use of two sequence-specific primers. Degenerate primers (*DegTaqI*, *DegPstI*, *DegAluI* and *DegSacII*, including Q0) and the marker gene (*AphVIII*) specific primers (RB1 and RB4) are shown in Table 1.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Outline of the principles for RESDA PCR. This schematic is adapted from González-Ballester *et al.*, 2005.</figcaption></figure>

<table><caption>**Table 1.** Primers and their sequences used in this protocol</caption><thead><tr><th>Primer nature</th><th>Primer name</th><th>Primer sequences (5'-3')</th></tr></thead><tbody><tr><td rowspan="4">Degenerate</td><td>DegTaqI</td><td>5'-CCAGTGAGCAGAGTGACGIIIIINNSWGTCGAA-3'*</td></tr><tr><td>DegPstI</td><td>5'-CCAGTGAGCAGAGTGACGIIIIINNSCTGCAGW-3'</td></tr><tr><td>DegAluI</td><td>5'-CCAGTGAGCAGAGTGACGIIIIINNSWCAGCTT-3'</td></tr><tr><td>DegSacII</td><td>5'-CCAGTGAGCAGAGTGACGIIIIINNSCCGCGGW-3'</td></tr><tr><td rowspan="3">Specific primers</td><td>RB1*</td><td>5'-AGCTGGCCACGAGGAGGAC-3'</td></tr><tr><td>RB4*</td><td>5'-TGGTTCGGGCCGGAGTGTTTC-3'</td></tr><tr><td>Q0</td><td>5'-CCAGTGAGCAGAGTGACG-3'</td></tr></tbody></table>
*I, inosine, N, A + T + G + C; S, G + C; W, A + T.

*Gene-specific primers (RB1 and RB4) can be designed based on the type of marker gene (i.e., *AphVII*, *Ble* and *aadA*) used for mutagenesis.

1. The first amplification

In the 1st amplification, the *AphVIII* gene-specific primer (RB1) and one of the degenerate primers are used to amplify a fragment flanking the antibiotic marker gene *AphVIII* from the genomic DNA purified from the mutant. Four independent reactions can be set up by using four different degenerate primers and RB1, respectively. The composition of the PCR reaction mixture and cycling program are shown in Table 2 and Table 3, respectively.

<table><caption>Table 2. Composition of the PCR reaction mixture used for the 1st amplification</caption><thead><tr><th>Component</th><th>Volume (µl)</th></tr></thead><tbody><tr><td>10x Standard Taq Reaction Buffer</td><td>2.0</td></tr><tr><td>dNTPs (2.5 mM each)</td><td>1.6</td></tr><tr><td>Taq DNA polymerase (5,000 U ml<sup>-1</sup>)</td><td>0.2</td></tr><tr><td>*Degenerate primer (10 µM)</td><td>0.2</td></tr><tr><td>RB1 (10 µM)</td><td>0.2</td></tr><tr><td>10% DMSO</td><td>1.0</td></tr><tr><td>Genomic DNA (50 ng µl<sup>-1</sup>)</td><td>4.0</td></tr><tr><td>H<sub>2</sub>O</td><td>10.8</td></tr><tr><td><strong>Total volume</strong></td><td>20.0</td></tr></tbody></table>

*Use one of the degenerate primers first.

<table><caption>Table 3. The PCR conditions used for the 1st amplification</caption><thead><tr><th>Temperature and time</th><th>Cycles</th></tr></thead><tbody><tr><td>95 °C, 8 min</td><td>1</td></tr><tr><td>95 °C, 30 sec; 58 °C, 1 min; 68 °C, 3 min</td><td>5</td></tr><tr><td>95 °C, 30 sec; 25 °C, 3 min; 55 °C, 3 min; 68 °C, 3 min</td><td rowspan="4">20*</td></tr><tr><td>95 °C, 30 sec; 58 °C, 1 min; 68 °C, 3 min; 95 °C, 30 sec; 58 °C, 1 min</td></tr><tr><td>68 °C, 3 min; 95 °C, 30 sec; 40 °C, 1 min; 68 °C, 3 min</td></tr><tr><td>And finally 68 °C, 5 min</td></tr></tbody></table>

*Each cycle contains thirteen steps sequentially; and totally twenty cycles were set up.

2. The second amplification

To get more specific PCR products, the maker gene-specific primer (RB4) and the specific primer (Q0) at the 5' end of degenerate primers are used to amplify using the PCR from the 1st amplification as a template (One µl diluted PCR products (1/1,000) from 1st amplification). The composition of the PCR reaction mixture and cycling program are shown in Table 4 and Table 5, respectively.
<table><caption>Table 4. Composition of the PCR reaction mixture used for the 2nd amplification</caption><thead><tr><td>Component</td><td>Volume (µl)</td></tr></thead><tbody><tr><td>2x Xtreme Buffer</td><td>10.0</td></tr><tr><td>dNTPs (2.5 mM each)</td><td>3.2</td></tr><tr><td>KOD Xtreme Hot Start DNA polymerase</td><td>0.2</td></tr><tr><td>Q0 (10 µM)</td><td>0.4</td></tr><tr><td>RB4 (10 µM)</td><td>0.4</td></tr><tr><td>Template (diluted 1st PCR product, 1/1,000)</td><td>2.0</td></tr><tr><td>H₂O</td><td>3.8</td></tr><tr><td>Total</td><td>20.0</td></tr></tbody></table>

<table><caption>Table 5. The conditions used for the 2nd PCR amplification</caption><thead><tr><td>Temperature and time</td><td>Cycles</td></tr></thead><tbody><tr><td>94 °C, 2 min</td><td>1</td></tr><tr><td>98 °C, 10 sec; 60 °C, 30 sec; 68 °C, 2 min</td><td>32</td></tr><tr><td>4 °C</td><td></td></tr></tbody></table>

### C. Agarose-gel electrophoresis and DNA fragments purification

1. Separate the PCR products from the 2nd amplification on 1.0% agarose gel at 100 V for 30 min by electrophoresis, and then stain the gel with SYBR Safe (1/1,000) and take image with a GelDocXR System (Bio-Rad Laboratories). Some representative results are shown in Figure 2, and the examples of RESDA-PCR success rate after amplification by four independent degenerate primers and RB1, followed by further amplification with RB4 and Q0 primers are shown in Table 6.

2. Cut-off the well-separated PCR products (marked with *) from the gel and purify using the NucleoSpin Gel and PCR Clean-up Kit (MACHEREY-NAGEL) according to manufacturer's instructions.

<figure><img src="image_3.png" /><figcaption>Figure 2. Examples of results of RESDA-PCR using the degenerate primer DegTaqI. The PCR products shown are the results of two rounds of PCR reactions: using DegTaqI and RB1, followed by further amplification with RB4 and Q0 primers.</figcaption></figure>
Note: M refers to DNA marker (Exact Ladder DNA PreMix 2 log); and each lane (1, 2, 3...) refers to the amplification from independent single mutant. The black asterisk indicates the PCR fragment that will be cloned before sequencing, and the red asterisk indicates the PCR fragment that can be directly sequenced.

<table><caption>Table 6. Examples of RESDA-PCR success rate after amplification with four independent degenerat primers and RB1, followed by further amplification with RB4 and Q0 primers</caption><thead><tr><th>1st amplification primers</th><th>2nd amplification primers</th><th>Success amount</th></tr></thead><tbody><tr><td>DegTaqI and RB1</td><td>RB4 and Q0</td><td>8/16</td></tr><tr><td>DegAlul and RB1</td><td>RB4 and Q0</td><td>2/16</td></tr><tr><td>DegPstI and RB1</td><td>RB4 and Q0</td><td>8/16</td></tr><tr><td>DegSacII and RB1</td><td>RB4 and Q0</td><td>3/16</td></tr></tbody></table>

### D. Subsequent cloning of DNA fragment

Since the recovered PCR products generated by proofreading polymerases (KOD Xtreme Hot Start DNA polymerase) are blunt-end, in this protocol pCR II-Blunt-TOPO vector is used. Other Taq DNA polymerase can also be used in this step.

1. Directly ligate the recovered fragments to pCR II-Blunt-TOPO vector. This step can be completed within 5 min without adding a single deoxyadenosine (A) to the 3' ends of PCR products. Add the ligation reaction components into a tube as described in Table 7, mix gently and incubate for 5 min at 22 °C.

Note: pCR-Blunt II-TOPO allows direct selection of recombinants via disruption of the lethal E. coli gene, ccdB (Bernard et al., 1994). Cells that contain a non-recombinant vector are killed upon plating.

2. The ligated products are transformed into One Shot competent cells (Thermo Fisher Scientific) according to manufacturers' instructions. Transformants are selected on LB agar plates containing 50 µg ml⁻¹ kanamycin (37 °C for overnight).

<table><caption>Table 7. Setup of the cloning reaction using the Zero Blunt™ TOPO™ PCR cloning kit</caption><thead><tr><th>Component</th><th>Volume (µl)</th></tr></thead><tbody><tr><td>PCR product</td><td>4.0</td></tr><tr><td>Salt solution</td><td>1.0</td></tr><tr><td>pCR II-Blunt-TOPO vector</td><td>1.0</td></tr><tr><td>Total volume</td><td>6.0</td></tr></tbody></table>

### E. Sequencing

1. For each PCR fragment, culture three independent colonies separately in LB medium and extract plasmids using NucleoSpin Plasmid (MACHEREY-NAGEL) following manufacturer's instructions.
2. Sequence the purified plasmids using the T7 universal primer, and example of sequencing results for one representative mutant is shown in Figure 3.

<figure><img src="image_4.png" /><figcaption>**Figure 3. Example of sequencing results for one representative mutant**</figcaption></figure>

Note: The partial sequences of AphVIII is underlined by red color and the sequences targeted to the genome of C. reinhardtii (v5.5) is underlined by black color, the sequences not underlined belong to the vector (pCR-Blunt II-TOPO) sequences.

## Data analysis

The obtained sequences were firstly blasted against *AphVIII* expression cassette to confirm whether the RESDA-PRC fragment contains sequences of this cassette. If it is so, the obtained sequences were subsequently blasted against the genome of *C. reinhardtii* (v5.5 at Phytozome) (Merchant *et al.*, 2007) to identify the flanking sequences around the cassette insertion site. The targeted genes of interest, *i.e.*, maintenance-type DNA methyltransferase and acyl-CoA oxidase gene, were further studied by molecular genetics and biochemical analysis as we previously described (Kong *et al.*, 2015 and 2017). All the experiments were performed in three biological replicates, and three plasmids of every single colony (containing PCR fragment) were sequenced, respectively. Statistical analysis was performed with SPSS program (version 19.0).

## Notes

1. This method is easily applicable to other insertional mutagenesis where marker genes other than *AphVIII* are used. Design of gene-specific primers is needed in this case.

2. In 1st PCR amplification, it is essential to use Taq DNA polymerase (*i.e.*, NEB) that has no proof-reading activity (lacking 3' to 5' DNase activity) to avoid the degradation of degenerate primers (containing inosine) hybridized to the genomic DNA. DMSO is used to reduce the hydrogen bonds in single-stranded DNA hairpin structures. We found out that the amplification with the DegPstI and DegTaqI degenerate primers had a higher probability of producing PCR products.

3. In 2nd amplification, the diluted PCR product from the 1st PCR reaction at 1/1,000 was found to be the best situation to have higher chances of amplification. The PCR product often ranges from 0.8 kb to 1.5 kb. If the PCR product is specific with sufficient amount, it can be recovered and sequenced directly, *i.e.*, without going through the step of cloning.

4. pCR II-Blunt-TOPO vector is alternative for subsequent cloning of DNA fragment; it is also possible to use universal TA Cloning techniques for sub-cloning and DNA sequencing.
## Recipes

1. 0.7 M NaCl

Add 40.95 g NaCl crystalline (Sigma-Aldrich) to 800 ml sterilized MilliQ water and stir until NaCl is dissolved, and make the final volume to 1 L

2. 10% SDS

Dilute SDS powder (Sigma-Aldrich) with sterilized MilliQ water (1:10, w/v) to make the working solution

3. 0.5 M Tris-HCl, pH 8.0

Add 60.55 g Tris base power (Sigma-Aldrich) to 800 ml sterilized MilliQ water and stir until Tris base is dissolved, adjust the pH to 8.0 with HCl using pH meter to monitor the pH change and make the final volume to 1 L

4. 10 mM EDTA

Add 2.92 g Tris base power (Sigma-Aldrich) to 800 ml sterilized MilliQ water and stir until EDTA is dissolved and make the final volume to 1 L

5. Lysis buffer

Dilute 0.5 M Tris-HCl, pH 8.0, zss10 mM EDTA, 0.7 M NaCl, 10% SDS and Proteinase K (20.0 mg ml⁻¹) to a mixture of the final concentration (100 mM Tris-HCl, pH 8.0, 1.75 mM EDTA, 150 mM NaCl, 2% SDS, and 1.0 mg ml⁻¹ Proteinase K)

6. 5 M KCl

Add 372.76 g KCl crystalline (Sigma-Aldrich) to 800 ml sterilized MilliQ water and stir until KCl is dissolved, and make the final volume to 1 L

7. 10% CTAB

Dissolve CTAB powder (Sigma-Aldrich) with 0.7 M NaCl (10:100, w/v)

8. 10% DMSO

Dilute the DMSO (Sigma-Aldrich) with PCR-grade H₂O (1:10, v/v) to the working solution

9. 1.0% agarose gel

Add agarose (Sigma-Aldrich) to 1 x TAE (1:100, w/v)

10. 1x TAE

Dilute UltraPure 10x TAE buffer (Thermo Fisher Scientific, UltraPure™) with sterilized MilliQ water (1:10, v/v) to make the working solution

11. 70% ethanol

Dilute Ethanol (Sigma-Aldrich) with sterilized MilliQ water (7:3, v/v) to make the working solution

## Acknowledgments

The work was supported by the French Agence Nationale pour la Recherche (ANR-Diesalg and ANR-MUsCA). This protocol is based on the protocol published by González-Ballester *et al.* (2005). There are no conflicts of interest.
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