2.2 AFLP-PCR

AFLP cocktail (recipe is for one rxn)

0.5 µL labeled EcoRI-primer (@ 10 ng/µL)
0.6 µL unlabeled MseI-primer‡ (@ 50 ng/µL)
0.8 µL 5 mM dNTPs
2.0 µL 10x-Promega Taq buffer (Mg-free)
1.2 µL 25 mM MgCl2
0.08 µL (0.4 U) Taq Pol (Promega)
13.82 µL dH20

‡ The basic MseI-primer is:

5'-GAT GAG TCC TGA GTA Axy z-3' 

like with the EcoRI-primer, x, y, and z represent selective bases that are specific and constant within a primer but vary between primers. [We typically use 2 bp selective extensions in our MseI-primers. The Zabeau group, as well as commercially available kits use 3-bp selective extensions in combination with 1- bp selective preamplification (see 2.0, Note 3) ]

PCR reactions (recipe is for one rxn)

1 µL preamplified DNAi
19 µL PCR cocktail

- amplify using "AFLP" as listed below:

"AFLP" thermocycle profile

1) 94° C, 2 min
2) 94° C, 30 sec
3) 65° C, 30 sec, -0.7° C per cycle starting next cycle
4) 72° C, 1 min
5) repeat steps 2 to 4, 11 times
6) 94° C, 30 sec
7) 56° C, 30 sec
8) 72° C, 1 min
9) repeat steps 6 to 8, 25 times
10) 72° C, 2 min
11) hold @ 4° C

3.0 PAGE of PCR products

Sample preparation

1) Mix each rxn w/ an equal vol of 2x formamide buffer (98% formamide, 10 mM EDTA, pH 8.0, plus bromophenol blue & xylene cyanol for tracking dye).

2) Incubate samples @ 90° C for ~ 45 min [this reduces the vol and thus increases the specific activity of the samples].

Electrophoresis

1) Load sample on gel [5.0% acrylamide/bisacrylamide (19:1), 7.5 M urea, 1x TBE§ and run @ 40-50 W (constant wattage) [40-50 V/cm, like for sequencing gels] with 1x TBE as the running buffer.

2) Run samples until the xylene cyanol is about 2-3 cm from the bottom of the gel and stop. [When 1x TBE is used in the gel and running buffer, and the gel is electrophoresed @ 50 W, it takes about 3.5-4 h for the xylene cyanol to get to the indicated position.]

Fixing & autoradiography

1) Fix gel in 10% acetic acid for 30 min.

2) Dry gel onto filter paper (E&K Scientific Products, cat# 2388-4567, 46 cm x 57 cm sheets) for _ 1 hr w/ heat.

3) Generate autoradiograph using Kodak BioMax film (or like product from another supplier). [This film gives nice O/N exposures when using either 33P or 32P as the label.]

4.0 Linkage analysis

Analyze F2 (or F3) recombinants relative to the two parent ecotypes (WT1, the genetic background of the mutant; and WT2, the polymorphic wild-type to which mutants were outcrossed, see Sec. 1.0) used as the backgrounds to generate the recombinants. [We typically use Columbia as the parental stocks (WT1 line) for mutagenesis and Landsberg erecta as our WT2 line.] Linkage is assessed by finding polymorphic bands specific to the WT2 parent that are missing in a large sample of F2 individuals (see below for suggested population size). The reason for looking for the absence of a WT2-specific band rather than the presence of a WT1 band is the inability to discern heterozygotes from homozygotes. For example, any F2 individual having a WT1-specific band could be either linked at one chromosome or both. The absence of a WT2-specific band requires therefore that the DNA specified by that polymorphic marker is homozygous for WT1 DNA in the F2 recombinant; or both chromosomes contain WT1 DNA that is linked to the locus of interest. The pitfall of this approach is that the presence of a WT2-specific band in an F2 individual can therefore be either WT2/WT1 or WT2/WT2. For example, if 1 in 100 F2 individuals has a WT2-specific band of interest the recombination percentage (linkage) can be either 0.5% (1 chromosome in 200) or 1% (2 chromosomes in 200). We therefore suggest examining at least 400-500 chromosomes to have high confidence in the linkage results. Any WT2-specific bands of interest are then subcloned and used to generate linked DNA fragments and oligos for library screening as described below.

4.1 Band isolation and subcloning

Band isolation

1) Perform a standard AFLP-PCR rxn with the WT1 and WT2 parents using the primer pair(s) (EcoRI-primer should be end labeled) that result in an excluded WT2 band(s) in the F2s

2) Precipitate the PCR products. Adjust the rxn products to 0.3 M Na-acetate and add 2.5 vol EtOH, incubate @ -20° C for _ 20 min, then spin @ 12K x g for 5 min. Resuspend each DNA in just enough 2x formamide buffer to load into 1 lane on a standard PA-AFLP gel. Run gel, dry (DO NOT FIX), and generate an autorad as per normal (see Sec. 3.0).

3) Cut out band of interest from dried gel (using autorad as the guide) and isolate the DNA via the "crush & soak" method of Sambrook et al. (Vol 1, pp. 6.46-6.48). Precipitate the DNA and resuspend in 10 µL of TE (pH 7.5). Use this DNA as the template for PCR amplification with "Universal AFLP Cloning"-primers (U-EcoRI and U-MseI) as described below.

PCR for subcloning

- Set up PCR rxns as per Sec. 2.0 except that the PCR cocktail contains 0.5 µL of each U-EcoRI- and U-MseI-oligos£ and only 13.92 µL dH2O instead of 14.42 µL per rxn. Use 1 µL of the DNA in step 3 above per rxn. Amplify with "PRE-AFLP".

£ U-EcoRI- and U-MseI-oligos are as follows: 

               U-EcoRI = 5'-GCG GAA TTC CGT AGA CTG CGT ACC AAT TC-3' 

                     - this oligo has an EcoRI site in its 5' end - 

               U-MseI = 5'-GCG CTG CAG GAC GAT GAG TCC TGA GTA A-3' 

                     - this oligo has a PstI site in its 5' end - 

Subcloning

1) Digest a desired amount of above PCR product with EcoRI and PstI, and ligate into like restricted plasmid vector of choice. [NOTE: There is some probability that the fragment of interest contains a PstI site. Cloner beware!]

2) Transform E. coli with recombinant plasmid and select for plasmid+ colonies by standard methods.

[NOTE: We typically use a two step verification of marker clones (as described below); one based on PCR and one based on Southern blot analysis.]

Clone verification I: Colony PCR

1) Pick plasmid+-selected colonies w/ toothpick

2) Dot onto new LB plate plus selective antibiotic

3) Inoculate 10-25 µL of digestion buffer (see below) with remaining cells on toothpick

Digestion Buffer
8.9 vol TE, pH 8.0
1.0 vol 10% (w/v) Laureth-12
0.1 vol 20 mg/mL Proteinase K

4) Incubate @ 55° C, 2 hrs

5) Heat inactivate Proteinase K @ 95° C, 10 min

6) Use 1 µL of digestion for PCR

7) Amplify as per AFLP-PCR (Sec. 2.2) using the EcoRI- (end-labeled) and MseI-primers that were used in step 1 of Sec. 4.1 (Do not pre-amplify! Just use DNA from digestion directly.). Adjust the volume of PCR cocktail such that the total rxn (cocktail + template) is 20 µL. Also amplify both WT1 and WT2 parents and any recombinants that have the WT2-specific band.

8) Compare banding patterns of amplified E. coli DNA (plasmids) with that of the WT1 & 2 parents and any F2 recombinants, and identify putative clones with the expected mobility.

9) Pick colonies for 4-6 putative clones from the plate generated in step 2 above and generate O/N cultures for plasmid isolation and Southern blot analysis (see below).

Clone verification II: Southern

1) Isolate plasmid DNA (by any standard technique) from O/N cultures of clones selected in step 9 above.

2) PCR label plasmid DNA by linear amplification using the MseI primer (used in step 1 of Sec. 4.1) as described below.

Single strand PCR labeling (recipe is for a single labeling rxn)
3 µL MseI-primer (@ 50 ng/µL)
1.0 µL 0.5 mM dATP, dGTP, dTTP
15.0 µL a32P-dCTP (3000 Ci/mmol)
2.5 µL 10x-Promega Taq buffer
1.5 µL MgCl2
0.5 µL Taq Pol
1.0 µL dH2O
1.0 µL ds plasmid DNA (25 ng/µL)

- amplify using "PRE-AFLP" theromcycle profile

3) ElectroBlot a standard AFLP gel (according to standard methods eg. Ausubel et al Current Protocols in Molecular Biology)of WT1 & WT2 parents, any recombinants having the WT2-specific band, and a few non-recombinants F2s (ones not having the WT2-specific band; as controls), that have been amplified as per Sec. 2.0-2.2 using the EcoRI- (33P end-labeled) and MseI-primers that were used in step 1 of Sec. 4.1.

4) Hybridize w/ labeled plasmid by standard methods.

[WT2-specific clones can be unambiguously determined from an autorad of the hybridized blot.]

- Cloned AFLP fragments can be used in a variety of manners as tools to screen various nucleic acid libraries (YAC, BAC, P1, cosmid, lambda, cDNA), a couple of examples are given below: -

Sequence based probes

1) Sequence cloned AFLP fragment(s) by standard methods.

2) Based on sequence, generate oligos to sites internal to the EcoRI and MseI adapter sequences (plant specific sequences).

3) Use these oligos to screen various libraries via PCR.

Insert based probes

- Use the insert or whole plasmid containing the of interest in a variety of ways -

1) Label ds insert DNA that has been liberated from vector DNA by standard RE digestion. Use labeled DNA in hybridizations for filter based library screening by standard techniques.

2) Label whole, linearized ds plasmid containing the insert and use it for screening filters as above in step 1.

3) Use oligos generated in step 2 of Sequence based probes above to make single stranded insert DNA probes. This can be accomplished as described in step 2 of the Clone verification II section of Sec. 4.2, with the exception that both insert-specific oligos are used as primers instead of an MseI-primer (adjust total vol appropriately). Use this labeled DNA for screening filter as described in step 1 above.

Acknowledgments

We wish to thank Dr. Chris Somerville for introducing us to the existence of AFLP technology and for his continued support and help throughout our work. We also wish to thank Dr. Shuana Somerville and members of her laboratory for many helpful discussions. Finally, we wish to thank Dr. Winslow Briggs who has embraced this technology and provided financial support for much of the AFLP work ongoing in the lab. We are also indebted to Glenn Ford for his assistance in providing this information in appropriate WWW formats.

Literature Cited

Liscum E and Briggs WR (1995) Mutations in the NPH1 locus of Arabidopsis disrupt the perception of phototropic stimuli. Plant Cell 7: 473-485.

Thomas CM, Vos P, Zabeau M, Jones DA, Norcott KA, Chadwick BP and Jones JDG (1995) Identification of amplified restriction fragment polymorphism (AFLP) markers tightly linked to the tomato Cf-9 gene for resistance to Claosporium fulvum. Plant J 8: 785-794.

Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M and Zabeau M (1995) AFLP: a new technique for DNA fingerprinting. Nucl. Acids Res. 23: 4407-4414.

Zabeau M (1992) European Patent Application, publication no. EP 0534858

Zabeau M and Vos P (1993) European Patent Application, publication no. EP 0534858.