AFLP Technologies

 

Our laboratory has used AFLP technology1 quite extensively and we felt strongly that others might benefit from our experiences with this technology. Hence, this page should serve as a useful reference tool for other researchers currently using or wishing to use AFLP technology.

1Disclaimer: AFLP technology was developed by Marc Zabeau and Pieter Vos (Zabeau M and Vos P, 1993, European Patent Application 92402629.7, 2; Vos et al., 1995) and rights are owned by Keygene N.V. (Agrobusiness Park 90, P.O. Box 216, NL-6700 AE Wageningen, The Netherlands) and Perkin-Elmer (Perkin-Elmer, Applied Biosystems, 850 Lincoln Centre Dr., Foster City, CA 94404, USA). We take absolutely no credit for the development of this technology, we merely wish to present some of our experiences with this technology.

 

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Uses of AFLP in Our Laboratory:

 

 

Chromosome Landing

A detailed description of the basic AFLP protocol we have used for "chromosome landing" studies of mutant genes in Arabidopsis thaliana can be found at my Carnegie Institution of Washington - DPB page. Although the CIW page provides the basic protocol we still use for "chromosome landing", several modifications have been made over the past couple of years. Some of these changes have recently been discussed elsewhere (Liscum, 1999; Liscum and Oeller, 1999), however many were not. Hence, we will attempt to discuss, in this section, modifications and changes that have been made to the basic CIW protocol.

Changes:

1) Sec. 2.0 of CIW protocol (Pre-amplification of template DNA). We no longer do a single primer preamplification of template DNAs. Instead we now do a two-primer, non-selective amplification using what we call the EcoRI-Core+adapt. and MseI-Core+adapt. oligos:

EcoRI-Core+adapt.

5'-CTC GTA GAC TGC GTA CCA ATT C

MseI-Core+adapt.

5'-GAC GAT GAG TCC TGA GTA A

The preamlification cocktail and PCR reaction are as described previously, except that 0.5 mL of the EcoRI-Core+adapt. and MseI-Core+adapt. oligos (@ 50 ng/mL) are used instead of the single EcoRI-oligo.1. The volume of H2O used is of course reduced by 0.5 mL to allow for the added oligo volume. The "PRE-AFLP" thermocycle profile has also been significantly modified as follows:

1)

94 C, 1'

2)

94 C, 30''

3)

56 C, 1'

4)

72 C, 1'

5)

repeat steps 2 to 4, 19 times

6)

72 C, 2'

7)

hold @ 4 C

All other notes and tips with regard to preamplifications remain applicable. We have found that the AFLP products arrising from DNAs preamplified according to the current method are not qualitatively different from those arrising from the CIW method. However, the quantitative efficiency of the current preamplication is considerably greater because of the use of two primers.

 

2) Sec. 2.2 of CIW protocol (AFLP-PCR). Everthing remains the same except that we now routinely scale down the reaction volume to 5.0 or 10.0 mL, from 20.0 mL. This saves enormously on reagents, which are essentially wasted since only 5-10% of the reaction is actually destined to be on a gel.

 

3) Sec. 3.0 of CIW protocol (PAGE of PCR products). We no longer incubate samples for ~45 min prior to gel loading. Currently samples are heated for 5-10 min to denature the samples only and then samples are loaded. It was found that the reduction in volume and increase in specific activity of the samples was not always desired. In cases where reaction products were very abundant or "dirty", such a volume reduction step resulted in autoradiographs that were often quite difficult to read.

Although we continue to use 1x TBE in our gel mix, we now use 0.5 x TBE in the running buffer. The banding seems not to be compromised, while reducing running times to 3 h.

 

4) Sec. 4.1 of CIW protocol (Band isolation and subcloning). Several modifications and improvments have been made to this section of our original protocol. We hope to have a summary of improvements up here soon.

Genotyping and "Ecotyping"

 

 

 

 

 

 

 

 

 

 

 

Studies of Differential Gene Expression

 

 

 

 

 


References

Liscum E (1999) AFLP: Studies on plant development. In PCR Methods Manual, M Innis, D Gelfand, J Sninsky, eds (Academic Press, San Diego), pp. 505-519

Liscum E and Oeller PW (1999) AFLP: DNA fingerprinting to positional cloning. In Analytical Biotechnology: Genome Analysis, P Oefner, ed (CRC Press, Boca Raton), in press

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