AFLP Analysis of Genomic Variation in the Coast Redwood, Sequoia sempervirens

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Introduction to Sequoia sempervirens

Construction of cDNA Library

Results - My Progress So Far

Objective

Simpson Timber Company, located in Korbel, California, has an orchard which contains several cloned/transplanted redwoods from all over California. The Company owns a phenotypic profile for each of these trees, as they were chosen for their "ideal" lumber characteristics. The goal of this project is to find a correlation between the polymorphisms found with the AFLP (Amplified Fragment Length Polymorphism) procedure and the trees unique profile.

What is AFLP?

Procedure - AFLP Procedure Diagram

#1. Preparation of Plant Tissue & DNA Isolation

• Grind needles under liquid nitrogen conditions with a stainless steel mortar and pestle
• Put ground tissue powder in a 'bead-beater' tube that is 1/3 filled with Zirconia beads
• Add buffer (detergent) and RNase (enzyme) and beat in Mini Bead-Beater.
• Centrifuge out beads and plant tissue; remove supernatant
• Incubation @ 65oC --> lyses cells
• DNA isolation is done with the DNeasy Plant Mini Kit from Qiagen

#2. Restriction Digest

• Genomic DNA is digested with 2 restriction enzymes: EcoR 1 (having a 6 bp recognition site) and Mse 1 (having a 4 bp recognition site).
• A large number of fragments are generated; EcoR 1-Mse 1 fragments are preferentially amplified (rather than EcoR 1-EcoR 1 or Mse 1-Mse 1 fragments)

#3. Adapter Ligation

• Genomic DNA fragments are ligated to EcoR 1 and Mse 1 adapters to generate template DNA for amplification
• These adapter sequences serve as primer-binding sites on the restriction fragments... therefore, it is possible to amplify many DNA fragments without having prior sequence knowledge.

#4. PCR 1 - pre-amplification

• purpose - to increase the amount of template available for mapping
• genomic DNA's are amplified with AFLP primers, each having two selective nucleotides (E-AC and M-CC)

#5. PCR 2 - final amplification

• products from pre-amp are used as template for selective amplification using 2 AFLP primers - a fluorescently labeled E-primer with 3 selective nucleotides (E*-ACA or E*-ACG) and an M-primer with 4 selective nucleotides (M-CCAC, M-CCAG, or M-CCAT)

#6. Gel Analysis

• Products from the selective amplification are separated on an 8% denaturing polyacrylamide gel; run on LiCor Gel Analyzer.
• The resultant banding pattern (fingerprint) can by analyzed for polymorphisms.
• click here to see gel "sizing standard" (ladder ; 50-700bp)