AFLP Protocol

NOTE: The protocol presented below is based on the Amplified Fragment Length Polymorphism (AFLP) technology developed by Marc Zabeau and colleagues at Keygene N.V., Agrobusiness Park 90, P.O. Box 216, NL-6700 AE Wageningen, Netherlands (Zabeau, 1992; Zabeau and Vos, 1993; Vos et al., 1995). The AFLP technology is covered by patents and patent applications owned by Keygene N.V. Both, Life Technologies (Gathersberg, MD, USA) and Perkin Elmer (Applied Biosystems Division, Foster City, CA, USA) market research kits (under license) for AFLP fingerprinting of plant DNAs.

 

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Background Rational

AFLP as developed by Keygene, was designed as a highly sensitive method for DNA fingerprinting to be used in a variety of fields, including plant and animal breeding, medical diagnostics, forensic analysis and microbial typing, to name a few. We are using this technology to generate DNA based markers for cloning genes involved in phototropic responses in higher plants that have only been identified genetically by mutant phenotype(s) (see Liscum and Briggs, 1995). We have had tremendous success to date using the technology decribed below (see pg. 4). (Recently, Thomas et al. (1995) reported the use of AFLP technology in the identification of tightly linked markers flanking (within 15.5 Kb) the Cf-9 resistance gene of tomato. Although the Cf-9 gene had been previously isolated via transposon tagging the rapid identification of markers within 15.5 Kb of the locus provide support for the contention that AFLP technology can be exploited for gene isolation by positional cloning.) The "guts" of how AFLP works is summarized in the following paragraphs.

The power of AFLP is based upon the molecular genetic variations that exist between closely related species, varieties or cultivars. These variations in DNA sequence are exploited by the AFLP technology such that "fingerprints" of particular genotypes can be routinely generated. These "fingerprints" are simply RFLPs visualized by selective PCR amplification of DNA restriction fragments. In order to give a brief summary of the working theory behind AFLP we will describe the technique as we use it to generate AFLPs between two Arabidopsis ecotypes, Columbia and Landsberg erecta. 1) Genomic DNA from each ecotype is digested to completion with two restriction enzymes, one EcoRI (having a 6 bp recognition site) and the other MseI (having a 4 bp recognition site). EcoRI cuts ~ every 2-2.5 Kb in Arabidopsis, while MseI cuts ~ every 300-400 bp. Thus a large number of fragments are generated with relative abundances as such: MseI-MseI >>> MseI-EcoRI >>> EcoRI-EcoRI. As discussed below we target the MseI-EcoRI and EcoRI-EcoRI fragments for analysis. 2) Specific ds oligonucleotide adapters (~ 25-30 bp) are ligated to the restricted DNA fragments. 3) Oligos homologous to the adapters, but having extensions at the 3'-end are used to amplify a subset of the DNA fragments. These extensions can vary in length from 1 to 3 bp, but are of defined length for a given primer. The sequence of the extension can also vary from one primer to another but is of a single, defined sequence within a given primer. The selective nature of AFLP-PCR is based on the 3' extensions on the oligonucleotide primers. Since these extensions are not homologous to adapter sequence, only plant DNA fragments complementary to the extensions will be amplified due to the inability of Taq DNA polymerase, unlike some other DNA polymerases, to extend DNAs if mismatches occur at the 3'-end of a molecule that is being synthesized. Therefore only a subset of the entire genome is amplified in any reaction. For example, if 2 bp extensions are used only one in 256 molecules is amplified assuming Taq polymerase cannot tolerate mismatches as discussed above. However, since MseI-MseI fragments predominate the population of fragments to be amplified, we need to further limit the number of fragments that are actually visualized so that a manageable number is observed. We do this by labeling the primer directed against the EcoRI-adapter sequence since MseI-EcoRI and EcoRI-EcoRI fragments will be a more limiting subset of the total DNA fragments. Finally, the amplified DNAs are separated on a polyacrylamide gel (sequencing type) and an autoradiograph is generated.

By labeling the EcoRI-directed primer and using 2 bp extensions on both primers we typically observed 100-200 bands on the autorad from any given primer pair. Only a subset of these total bands are polymorphic between two related individuals, such as Arabidopsis Columbia and Landsberg erecta ecotypes. Any given AFLP primer pair generates on average 10-20 ecotype specific bands (conservatively), thus given 256 possible primer pair combinations when using 2 bp selective 3' ends, 2500-5000 markers can be generated quite rapidly. Given a genome size of 100 Mbp for Arabidopsis, and assuming equal recombination frequencies over the entire genome (or equal distribution of markers throughout the genome), generation of 2500 markers would place any pair of markers within 40 Kb of each other, on average. This level of physical linkage would result is the generation of markers that are _ 0.3 cM (0.3% recombination) within the mutant locus of interest. Thus AFLP should eliminate the "walk" typically associated with non-T-DNA-tagged mutants, and substitute the "hop" or "land".

 

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AFLP Protocol (Abridged Version 1.3, 12/95)

1.0 Generate polymorphic recombinant F2 (or F3) population

Outcross desired mutant in one genetic background (parental ecotype of the mutant hence referred to as WT1) to a wild-type plant of another genetic background (WT2) known to be molecular genetically polymorphic to WT1. [We have used three WT ecotypes of Arabidopsis for our work (Columbia, Landsberg erecta, & WS) and each is about equally polymorphic from any other. There is no reason to believe that other ecotypes would not be equally suitable.] Self the resultant F1 seed and select homozygous mutants from the subsequent F2 generation. Collect tissue from homozygous mutant F2 plants, as well as WT1 and WT2 plants, for DNA isolation as described below in Sec. 1.1 . Self homozygous mutant F2s and collect seed for future use. It is also helpful to save a few non-mutant F2s for tissue collection and F3 seed collection as they may be useful controls for future experiments.

1.1 Isolate genomic DNA

NOTE: Use any mini(micro)prep that yields good quality DNA. CsCl-purified DNA is not necessary. The main contaminants of concern are carbohydrates. If the A260/A230 is _ 2.2 the nucleic acid should be of adequate quality. Dark adapting the plants for 2-3 d prior to tissue harvest is usually enough to insure low carbohydrate content.

 

Microprep

1.) Dark adapt 3-4 week old plants (just beginning to bolt) for 2-3 d, then harvest tissue (1-4 rosette leaves) into 1.5 mL microfuge tubes w/ liquid N2. Grind tissue on liquid N2 with pellet pestle (Kontes Scientific Glassware/Instruments; cat #749520-9001 for pestles only, #749520-0000 for tubes and pestles). [Using an electric drill to hold and rotate the pestle works extremely well!] Hold ground tissue at _ -80° C until extraction.

2.) For extraction of nucleic acids, add 750 µL extraction buffer (see below) to ground tissue, vortex to mix thoroughly, and incubate @ 65° C for 10 min.

 

Extraction Buffer
50 mM Tris, pH 8.0
10 mM EDTA, pH 8.0
100 mM NaCl
1.0 % (w/v) SDS
10 mM ß-mercaptoethanol (add just before use)

3) Add 150 µL 5 M K-acetate, vortex, incubate on ice for 20 min. Spin @ 12K x g in microfuge for 10 min.

4) Transfer 750-800 µL supernatant to new microfuge tube and add an equal vol of isopropanol. Immediately spin down nucleic acids by centrifugation @ 12K x g for 1 min. [Some small pieces of tissue may pellet at this stage, but they will be removed in the next step.]

5) Wash pellet w/ 75% EtOH and resuspend in 200 µL H2O. Spin @ 12K x g , 2 min. Remove 180 µL of supernatant to new tube and add 20 µL 3 M Na-acetate (or K-acetate). Mix and add 500 µL EtOH, then incubate @ -20°C for _ 10 min. Pellet nucleic acids by centrifugation (12K x g, 5 mn). [This step will remove chlorophyll as well as any debris carried over from step 4.]

6) Wash pellet w/ 75% EtOH, dry and resuspend in 10-50 µL TE (pH 7.5).

7) Quantitate nucleic acids and assess purity (A260/A230; A260/A280). [This microprep yields nucleic acids with A260/A230 > 2.0 and A260/A280 ~ 2.0. Although most of the nucleic acid is probably RNA, in practice there appears to be a sufficient amount of DNA for reproducible AFLP results if one follows the protocols below.]

1.2 Restriction of DNA

 

(Recipe is for one sample)

0.5 µg genomic DNA (total nucleic acid from mini(micro)prep)
5 U EcoRI
5 U MseI
8.0 µL 5x-Pharmacia "One-Phor-All+" buffer [10x = 100 mM Tris-acetate, pH 7.5, 100 mM Mg-acetate, 500 mM K-acetate) w/ 250 ng/µg BSA (=5x OPA+- BSA)
Q.S. to 40 µL w/ dH20

- incubate @ 37° C for ~ 3 hrs

[Note: The digestion should not be done for significantly longer than 3 hrs, as it is necessary to have active enzymes present during the ligation step (Sec. 1.3 ) to ensure complete digestion and ligation]

1.3 Ligation of adapters

 

Adapter Preparation

 

EcoRI-adapter = 5'-CTCGTAGACTGCGTACC (EcoRI-oligo.1)

   CTGACGCATGGTTAA-5' (EcoRI-oligo.2)

- mix 1.7 µg EcoRI-oligo.1 and 1.5 µg EcoRI-oligo.2, 3 µl 10X One-Phor-All+,Q.S. to 60 µL w/ dH2O, heat to 95° C, and allow to cool to RT slowly. This gives a final concentration of 5 pmoles/µL and makes enough adapter for 60 ligations (see below).

 

MseI-adapter = 5'-GACGATGAGTCCTGAG (MseI-oligo.1)

     TACTCAGGACTCAT-5' (MseI-oligo.2)

- mix 16 µg MseI-oligo.1 and 14 µg MseI-oligo.2, 3 µl 10X One-Phor-All+,Q.S. to 60 µL w/ dH2O, heat to 95° C, and allow to cool to RT slowly. This gives a final concentration of 50 pmoles/µL and makes enough adapter for 60 ligations.

[Note: Adapter oligos should not be phosphylated, this prevents adater self ligation. Both adapters are engineered such that the ligation "kills" the restriction site to which the adapter is ligated.]

 

Ligation Rxn

Add 10 µL of following mix to each 40 µL digestion rxn:

(recipe is for one ligation rxn)

1.0 µL EcoRI-adapter
1.0 µL MseI-adapter
1.0 µL 10 mM ATP
4.0 µL 5x-OPA+-BSA
1 U T4 ligase (Pharmacia)
Q.S. to 10 µL w/ dH20

- Incubate @ 37° C for 3 hrs to O/N

2.0 Pre-amplification of template DNA

[Note 1: This pre-amplification step helps to "clean up" some background noise that is observed on autorads of gels where non-preamplified DNA was used as a template. Furthermore, for every 1 µL of total ligated nucleic acid that is preamplified one will have 150 µL of template for further amplification (see below).]

[Note 2: For this and subsequent PCR we use an MJ Research thermocycler (# PTC100; Watertown, MA, 1-800-729-2165). This thermocycler accepts 96-well microtiter plates, which is a more efficient way to amplify the multiple samples required for AFLP.]

[Note 3: The AFLP procedure reported in NAR by Vos et al. (1995) uses a preamplification with both EcoRI and MseI primers having 1-bp, 3'-extensions, followed by AFLP-PCR with primers having 3-bp, 3'-extensions, whereas our preamplification uses only our "EcoRI-oligo.1" (adapter oligo with no extension) (see below), and our AFLP-PCR uses only 2-bp, 3'-extensions (see 2.2). Life Technology and Perkin Elmer protocols use the Vos et al. (1995) preamplification and AFLP-PCR. In our experience the single primer amplification (EcoRI-oligo.1) works quite well in comparison to the 1-bp two primer preamplification without reducing the number of polymorphic bands in the subsequent AFLP-PCR reactions. However, it should be noted that there is alot of room for "personalizing" the preamplification and AFLP-PCR steps to fit ones needs.]

 

Pre-amplification cocktail (recipe is for one rxn)

0.5 µL EcoRI-oligo.1 (@ 50 ng/µL)
0.8 µL 5 mM dNTPs
2.0 µL 10x-Promega Taq buffer [500 mM KCL, 100 mM Tris-HCl, pH 9.0 (@ 25° C), 1.0% Triton X-100; Mg-free)
1.2 µL 25 mM MgCl2
0.08 µL (0.4 U) Taq Pol (Promega)
14.42 µL dH2O

PCR reactions (recipe is per rxn)

1 µL cut/ligated nucleic acid (from above) [equals 10 ng total nucleic acid)
19 µL pre-amplification cocktail

- amplify using "PRE-AFLP" as listed below:

"PRE-AFLP" thermocycle profile

1) 94° C, 2 min
2) 94° C, 30 seci
3) 50° C, 30 sec
4) 72° C, 1 min
5) repeat steps 2 to 4, 34 times
6) hold @ 4° C

- Do NOT perform a "hot start".
-dilute PCR products 1:10 w/ TE (pH 7.5) and store @ 4° C (or -20° C for long term) -

2.1 Primer labeling (w/ 33P or 32P)

 

(recipe is for a 50 µL labeling rxn)§

10.0 µL EcoRI-primer† (@ 50 ng/µL)
10.0 µL g33P-ATP (or g32P-ATP) [3000 Ci/mmol]
5.0 µL 10x-OPA+ (no BSA)
1.0 µL (5-10 U/µL) T4 polynucleotide kinase (PNK)
24.0 µL dH2O

§50 µL labeling rxn makes enough "hot" primer for 100 PCR rxns.

† The basic EcoRI-primer is:

  5'-AGA CTG CGT ACC AAT TCx yz-3'

 

where, x, y, and z represent the selective bases on the 3' end of the oligo.

[We typically use only 2 bp selective extensions (x and y, no z in our primers). x, y, and z are specific and constant bases within a single primer but vary from primer to primer. Thus with 2 bp selective extensions one will have 16 independent primers.]

 

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