Bonjour à tous,
Je suis en train d'étudier une publication dans laquelle ils veulent créer de nouveaux marqueurs microsatellites chez l'arachide. Le protocole est décrit mais il y a une étape que je ne comprend pas bien.
Je vous mets le bout d'article en question (in english evidement):
Library construction Total genomic DNA libraries enriched for trinucleotide repeats were constructed following a standard protocol with minor modifications[49]. Peanut total genomic DNA was digested with five different enzymes (AluI, MseI, RsaI, Sau3AI, and Tsp509I) to identify which one pro- duced the most adequate fragment profile for library development. Fragments ranging in size from 200 to 800 bp were transferred to S&S NA 45 DEAE cellulose membranes, after electrophoresis in 1.5% low-melting agarose gel. DNA was resuspended in TE buffer and ligated to Tsp509 I adapters. Four genomic libraries enriched for trinucleotide repeats were constructed. Fragments were selected by hybridization with biotinylated trinucleotide repeats ((TTG)6,(TGG6, (ATG)6, (ATC)6) and recovered by magnetic beads linked to streptavidine. Enriched fractions were amplified by PCR, using a primer complementary to the adapters. Amplification products were purified using Wizard PCR Preps DNA purification system (Promega, Madison, WI), cloned into λ ZAP II phagemidvector (Stratagene) and then transformed into E. coli strain XL1-Blue. Transformed cells were grown on LB- Amp plates (50 μg/ml ampicillin) at 37°C, until plaques were between 0.5 and 1.0 mm in diameter. Individual clones were picked up from the plates and phage were eluted in SM buffer (500 μl) with adrop of chloroform. Clone DNAs were then amplified by PCR using primers complementary to the vector DNA flanking the insert (T3 or SK and T7 or KS primers,Stratagene, CA), and electrophoresed in 1.5% agarose gels in 0.5X TBE buffer to determine insert sizes. To determine the position of the SSRs relative to the vector cloning site, an anchored PCR strategy was performed for each positive clone [49], using T3 (or SK) and T7 (or KS) primers separately plus another primer complementary to the target SSR.
Premièrement, pourquoi passer par une étape de clonage ? C'est pour isoler chaque fragment ?
Ensuite, après cette étape de clonage, ils pratiquent une PCR ancrée. Quelle est le but de cette étape. Pourquoi ne pas passer directement au séquençage. Parce que si j'ai bien compris, cela va servir à ne garder que les fragments dans lesquels le microsatellite ne se trouve pas à une extremité. Mais c'est aussi visible par séquençage. Peut-être que la PCR coûte bien moins cher que le séquençage, et du coup ils ont préféré rajouter une étape ?
merci de votre aide
-----
