在通过<em>AphVIII</em>随机插入诱变产生的衣藻突变体中采用RESDA-PCR鉴定插入位点

Fantao Kong; Yonghua Li-Beisson

doi:10.21769/BioProtoc.2718

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Peer-reviewed

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

在通过AphVIII随机插入诱变产生的衣藻突变体中采用RESDA-PCR鉴定插入位点

Fantao Kong email

YL Yonghua Li-Beisson

发布: 2018年02月05日第8卷第3期 DOI: 10.21769/BioProtoc.2718 浏览次数: 7712

评审: Trinadh Venkata Satish TammanaAnonymous reviewer(s)

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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 (莱茵衣藻)

Insertional mutagenesis (插入诱变)

RESDA PCR (RESDA PCR)

Forward genetic (正向遗传)

Chlamydomonas mutant (衣藻突变体)

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

Falcon conical centrifuge tubes, 15 ml (Corning, catalog number: 430055 )
Eppendorf tube, 1.5 ml and 2.0 ml, Eppendorf Quality^TM(Eppendorf, catalog numbers: 0030120086 and 0030120094 )
Petri dishes, 90 mm in diameter (Thermo Fisher Scientific, Sterilin^TM, catalog number: 101R20 )
Sterilized toothpick (Fujian Fuhua, FUHUA FANGYUAN^TM, catalog number: 855 )
Chlamydomonas reinhardtii mutants generated by random insertional mutagenesis (Cagnon et al., 2013)
One Shot^TM TOP10 Chemically Competent cell (Thermo Fisher Scientific, catalog number: C404010 )
Zero Blunt^TM TOPO^TM PCR Cloning Kit (Thermo Fisher Scientific, catalog number: 450245 )
Taq DNA polymerase (5,000 U ml^-1), 10x Standard Taq Reaction Buffer, dNTPs (New England Biolabs, catalog number: M0273S )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
PCR-grade H₂O (Sigma-Aldrich, catalog number: W1754 )
Ethanol (Sigma-Aldrich, catalog number: 34852 )
KOD Xtreme Hot Start DNA polymerase, dNTPs (2 mM each), 2x Xtreme buffer (Merck, Novagen^®, catalog number: 71842 )
Agarose (Sigma-Aldrich, catalog number: A9539 )
ExactLadder^® DNA PreMix 2 log (OZYME, catalog number: OZYC002-500 )
SYBR^TM Safe DNA gel stain (Thermo Fisher Scientific, catalog number: S33102 )
UltraPure^TM 10x TAE buffer (Thermo Fisher Scientific, catalog number: 15558026 )
Luria broth (Sigma-Aldrich, catalog number: L1900 )
Kanamycin sulfate (Thermo Fisher Scientific, catalog number: 11815024 )
NucleoSpin^® Gel and PCR Clean-up Kit (MACHEREY-NAGEL, catalog number: 740609.10 )
NucleoSpin^® Plasmid Miniprep Kit (MACHEREY-NAGEL, catalog number: 740588.10 )
Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
Tris base (Sigma-Aldrich, catalog number: T6066 )
Hexadecyltrimethyl ammonium bromide (CTAB) (Sigma-Aldrich, catalog number: H9151 )
Isopropanol (Sigma-Aldrich, catalog number: W292907 )
Phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma-Aldrich, catalog number: P3803 )
Proteinase K (20.0 mg ml^-1) (Sigma-Aldrich, catalog number: P6556 )
Lysis buffer (see Recipes)
1. 0.7 M NaCl
2. 10% SDS
3. 0.5 M Tris-HCl, pH 8.0
4. 10 mM EDTA
5 M KCl (see Recipes)
10% CTAB (see Recipes)
10% DMSO (see Recipes)
1.0% agarose gel (see Recipes)
1x TAE (see Recipes)
70% ethanol (see Recipes)

Equipment

Shaker (Eppendorf, New Brunswick^TM, model: Innova^® 44 , catalog number: M1282-0002)
Accumet^TM pH meter (Fisher Scientific, model: 3-in-1 Set, catalog number: 13-636-AE153 )
PCR Thermal Cyclers (Thermo Fisher Scientific, Applied Biosystems^TM, model: 2720 , catalog number: ED000651)
Agarose electrophoresis tank (Bio-Rad Laboratories, model: Mini-Sub^® Cell GT, catalog number: 1704401 )
Gel Doc XR System (Bio-Rad Laboratories, model: Gel Doc^TM XR+ , catalog number: 5838)
Fisherbrand^TM Common bench-top vortexer (Fisher Scientific, catalog number: 02-216-125 )
Bench-top centrifuge (Beckman Coulter, model: Allegra^® 64R , catalog number: 367586)
Bench-top incubator (Eppendorf, New Brunswick^TM, model: S41i , catalog number: S41I230011)
NanoDrop 2000 (Thermo Fisher Scientific, model: NanoDrop^TM 2000 , catalog number: ND-2000)
Multisizer 3 Coulter counter (Beckman Coulter, model: Multisizer^TM 3 , catalog number: 6605697)

Software

SPSS program (version 19.0)

Procedure

English

中文翻译

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如何引用

Kong, F. and Li-Beisson, Y. (2018). Identification of Insertion Site by RESDA-PCR in Chlamydomonas Mutants Generated by AphVIII Random Insertional Mutagenesis. Bio-protocol 8(3): e2718. DOI: 10.21769/BioProtoc.2718.