AFLP initiators and adapters for Eco RI and MseI

Eco RI  Adapter:

Eco A1:     5’ - CTCGTAGACTGCGTACC

Eco A2:            CTGACGCATGGTTAA-5’

Selective initiators:

Mse I Adapter:

Mse A1:    5’-GACGATGAGTCCTGAG

Mse A2:        TACTCAGGACTCAT-5’

SOLUTIONS PREPARATION

*    Buffer PCR 10X: 

Keep at--20°C

*     dNTPs 5mM:

Add 200 µl of each of the nucleotides (dATP, dTTP, dCTP, dGTP)  to 3,200 µl of Milli-Q water ( or nucleases free water).  Aliquot in microcentrifuge tubes and keep at - 20°C.

*  Restriction Ligation Buffer 5X (RL 5X):

Keep at--20°C. 

*  1M Tris HAc pH 7.5: 

Weigh 6.055 g of Tris bases for 50 ml. Take to pH 7.5 with acetic acid, complete with Milli-Q water. 

Keep at 4ºC

*      ATP 10 mM

*      MgAc 1 M

*      KAc 1 M

*      DTT 250 mM

*      BSA 10 m g/ m l

*      Tris HCl 1M pH 8.3

Weigh 6.055 g of Tris base for 50 ml, take to pH 8.3 with HCL, complete to 50 ml with Milli-Q water. 

*      MgCl₂1 M

*      KCl 1M

*     Eco A and Mse A Adapters

     Prepare adapters according to the stock concentration. For example:

Eco A (5 mM):

 Mse A (50mM):

Primers: 

Prepare primers at 50 ng/µl concentracion.

TE 1.0 mM EDTA:

TE 0.1 mM EDTA:

Note: Use TE 0.1 mM EDTA to dilute the DNA and the primers.

 TBE 10X for PAGE:

1M Tris                                        

1M Boric Acid                                             

20 mM EDTA                                 

dd H₂O (complete to 1 L).

Loading Buffer for PAGE:

Xylene Cyanol                  11.2 mg

Urea                                 8.4 g

dd H₂O            Complete to 14 ml.

1 Kb ladder for PAGE:

1 Kb (1 m g// m l)              10 µl 

Loading buffer (PAGE)       130 µl

1X TBE (PAGE)                    60 µl 

Marker of 50 bp for PAGE:

50 bp (1 µg/µl)                  10 µl

Loading buffer (PAGE)       130 µl

1X TBE (PAGE)                    60 µl

 10% Ammonium persulfate 

 Sodium thiosulfate (10 mg/ml)

 Bind (for short glass plates):

Ethanol                            3 ml

Glacial acetic acid              9 µl

Bind silane                       9 µl

Others reagents needed to perform AFLP:

  References:                                          

 1.    Van der Lee, T., I. De Witte, A. Drenth, C. Alfonso, and F.Govers. 1997.  AFLP linkage map of the oomycete Phytophthora infestans.  Fungal Gen. and Biol. 21:278-291.

2.   Zabeau, M., P. Vos. 1993. Selective restriction amplification: a general method for DNA fingerprinting. European Patent Publication 92402629 (Publication No. EP0534858A1).                

POLYACRYLAMIDE GELS FOR DNA ELECTROPHORESIS  

Note: Denaturant polyacrylamide gels are utilized for sequencing of DNA and for the separation of AFLP products in electrophoresis equipment from BIORAD sequencing gels (Model Sequi Gen-GT 38x50 cm).  

1.       Preparation of sequencing plates:

The glass plates should be meticulously cleaned. Rinse the clean plates with de-ionized water to eliminate possible impurities, finally wash with ethanol .

  Short Glass Plate Preparation:

Treat the short glass plate with binding solution each time a gel is prepared.  

a)   Prepare fresh binding solution by adding 3 m l of bind silane to 1ml of 95% ethanol and 3 m l of acetic glacial acid.

  b)   Wipe a scrupulously cleaned plate using a KimWipes tissue saturated with 1 ml of freshly prepared binding solution. Make sure the plate is completely covered. You may use a tissue paper that does not leave  lint.

  c)   After 4-5 min, apply approximately 2 ml of 95% ethanol to the plate and wipe with a tissue paper in one direction and then perpendicularly to the first direction using gentle pressure. (Rubbing hard will remove an excessive amount of the bind silane, and the gel may not adhere as well).  

Repeat this operation three times, using a new tissue every time, to eliminate the excess of the adherent solution. This is important to prevent the binding solution from contaminating the long glass plate, which could result in a torn gel.

  Long Glass Plate Preparation:

  a)    Use new gloves before preparing the long plate to prevent cross-contamination with binding solution.

  b)    Wipe a scrupulously cleaned plate using a tissue saturated with Repel solution.

  c)   After 5-10 min, remove the excess of Repel solution by wiping the plate with a KimWipes tissue. Excess Repel may cause inhibition of staining.

  If the plate becomes contaminated with bind silane, soak it in 10% NaOH for 30-60 minutes.

2 .    Preparation of the sequencing gel:

a)    Wait until the glass plates dry completely (at least 30 min.).

b)    Assemble the glass plates before pouring the gel. There should be special care with filtration, uniform pressure of the clamps and combs and adequate spacers (use 0.4mm thick vinyl spacers and sharktooth comb).

  c)    Prepare the solution pre-mixture of 6% polyacrylamide (20:1, acrilamida:bisacrilamida),  500 ml:  

Store to 4ºC in a bottle covered with aluminum paper

d)   Prepare the gel as indicated below:  

      For one Gel:

      Pre-mixture of polyacrilamide        80 ml

      Temed                                            44 ul

      Ammonium persulfate 10%          440 ul    

e)   Pour the gel solution. Avoid bubble formation, but if they form, place the plates vertically and strike smoothly. Place carefully the combs along the plate. Allow the gel to polymerize horizontally at least for 2 hrs. Pre-run the gel to 1000 V by 15-30 min in order to balance it.

  f)   Place the samples. Run the gel at 500 V during the night or run a fast electrophoresis at 1600V for  5-6 hrs.

  g)    Stain with silver nitrate: Follow corresponding protocol.

Sequencing Gels Silver Stain

Reference:

Adapted from Promega protocols.  

Note: Silver stain is used for AFLP detection. The water and reagent quality have a great influence on stain success. Alternatively, the Promega stain kit (Catalogue No. Q4130) can be used.

 Protocol:  

1.   Solutions preparation:  

a)   Fix/stop solution (10% glacial acetic acid): add 200 ml of glacial acetic acid to 1,800 ml of ultrapure water (Milli-Q) or bi-distilled water.

  b)  Staining solution: dissolve 2 g of silver nitrate (AgNO₃) in 2 L of ultrapure water and add 3 ml of 37% formaldehyde.

 c)   Developing solution: dissolve 60 g of sodium carbonate (Na₂CO₃) in 2 L of ultrapure water. Chill to 10°C in an ice bath. Immediately before use, add 3 ml of 37% formaldehyde and 400 m l of sodium thiosulfate (10 mg/ml).

  2.  Separate the plates: after electrophoresis, carefully separate the glass plates using a plastic wedge. The gel should be strongly adhered to the short plate.

  3.   Fix the gel: place the gel in a plastic tray, cover it with the fix/stop solution and agitate it for 20 min or until the tracking dyes are not visible (This step is critical for DNA precipitation and the elimination of the urea).

  4.   Wash the gel: Rinse the gel twice using agitation in ultrapure water (2 min every time). Take out the plate from the tray and allow to drain for 10-20 seconds before placing it in the following washing.

  5.   Stain the gel: Transfer the gel to the stain solution and agitate for 30 min. Complete preparation of the developing solution. Pour the pre-chilled developing solution into a tray.

  Warning: The time of the next step (rinse) is very important. Prolonged rinses may cause a weak or lack of signal.

 6.   Rinse the gel: Submerge the gel briefly in the tray containing ultrapure water, drain and place the gel immediately into the tray of chilled developing solution. The time taken to submerge the gel in water and transfer it to the developing solution should not exceed 5-10 seconds.

 7.   Develop the gel: Agitate the gel until the bands begin to appear. Continue to develop until all bands are visible.  The developed bands appear fairly light. Prolonged development times results in high background.

 8.   Fix the gel: To finish the developing reaction, add stop solution (~ 500 ml) and leave in agitation for 2-3 min.  

 9.   Rinse the gel twice, in ultrapure water, 2 min each time.

10.  Dry the gel, leaving it at room temperature or using a conventional dryer.

11.  Permanent registries: The APC films are the most desirable format to make permanent the registries of the silver dyed gels. The APC films produce a positive and opposite image of the original on a white background. The gel should be completely dry before exposing the APC film. Handle the plates with gloves to avoid fingerprints.

  a)  Place the dry gel adhered to the plate (gel side up) on a white lightbox in dark room with the safelight on.  

b)   Place the APC film, emulsion side down, over the gel to be copied.

c)   Place a clean glass plate on the film to maintain contact between the gel and the film. Turn on the light box and expose the film for 1-2 min.  The exposure time depends of gel background or may vary with different light. Optimize the exposure time exposing strips of the APC film APC for several time intervals. In general, exposure times of 1-2 minutes produce good results.

  d)  Processing the exposed APC film as follows: Submerge in developing solution (Kodak GBX Developer) for 1-5 min,  wash during 1 min with de-ionized water, 3 min in fixer solution (Kodak GBX Fixer) and 1 min in de-ionized water.

PRINCIPLES:

AFLP is a highly sensitive method for fingerprinting genomic DNA of any origin and complexity. It has many potential applications such as monitoring the inheritance of agronomic traits in plant breeding, diagnostic of genetically inherited diseases, pedigree analysis, forensic typing, parentage analysis, etc. The AFLP technique has several advantages over other DNA fingerprinting systems. The most important are the capacity to examine an entire genome for polymorphism and its reproducibility. AFLP can be applied to any DNA sample including human, animal, plant and microbial DNA, giving it the potential to become a universal DNA fingerprinting system.

The AFLP technique has been developed by the company KeyGene (Wageningen, The Netherlands), which has filed property rights on this technology (Zabeau and Vos 1993).

AFLP is based on the selective PCR amplification of restriction fragments from a total digested genomic DNA.

• DNA that is being studied is digested with two different restriction enzymes: one that cuts frequently (the four-base restriction enzyme MseI) and one that cuts rarely (the six-base restriction enzyme EcoRI). Specific synthetic adapters for each restriction site are then ligated to the digested DNA (We perform the restriction and ligation steps in a single reaction).
• The ligated DNA is then subjected to a preliminary PCR amplification using oligonucleotide primers that are specific to the adapter/restriction sites. An extra nucleotide is added, for example A, thereby only a subset of the fragments of the mixture is amplified (those in which the restriction site sequence is followed directly by an A.
• A second amplification is then carried out using similar oligonucleotide primers, but with 2 extra bases (for example AC). Therefore, only a subset of the preliminary amplification reaction will undergo subsequent amplification during the second round of PCR (those in which the AC sequence follows the restriction site sequence).
• The subset of fragments is analyzed by denaturing polyacrylamide gel electrophoresis to generate a fingerprint and the DNA bands are detected by silver stain.
• The AFLP technique detects polymorphism due to changes in the MseI and

METHODOLOGY:

Before you start with AFLP fingerprinting, prepare all the necessary solutions that are indicated in Solutions preparation

1. Mix for restriction-ligation reaction

    a. Recipe is for one sample 

b. In a microcentrifuge tube add the restriction-ligation mixture and 10µl of DNA (500 ng in 10 µl). Total volume 50 m l. Note: Always prepare an additional volume of a reaction per every 20 reactions.

c. Incubate at 37°C for 4 hours.

d. Make a 1:4 dilution (add 150 µl of TE 0.1 mM EDTA)

e. Store at -20°C.

2. Mixture for pre-amplification reaction:

a. Recipe is for one sample

2. Add to PCR tube (0.2 ml):

5 µl of diluted DNA (digested and ligated) and 15 µl of pre-amplification mixture.

* Taq polimerase from Perkin Elmer (5U/µl) use 0.08 µl per reaction. Adjust the final volume with Milli-Q water

3.   Amplify using the following cycle profile:

             20 cycles at:

                         92°C for 60 sec

                         60°C for 30 sec

                         72°C  by 60 sec.

  For this and subsequent PCR we use an MJ Research thermocycler (# PTC 200).

4.   Bring up to 200 µl with TE 0.1mM EDTA the pre-amplified products.

5.   Store at 4°C (or -20°C for long term).

3.    Mixture for selective amplification:

             ddH₂Or                          10.9 µl       

             Eco (+2)                          0.5 µl      

             Mse (+2)                         0.6 µl

             DNTPs (5mM)                  0.8 µl

            PCR 10X                          2.0 µl

           Taq polymerase                 0.2 µl

                                                 15.0 µl             

a)    Add to a PCR tube:

5µl of pre-amplification product and 15µl of amplification mixture

b)   Conditions for amplification:

        Cycle 1:

              94°C           30 sec

              65°C           30 sec

              72°C           60 sec

         Cycle 2-13:

              94°C           30 sec

              65 - 0.7°C   every cycle 30 sec

              72°C           60 sec

         Cycle 14-36:

              94°C           30 sec

              56°C           30 sec

              72°C           60 sec

4 .    Electrophoretic run in denaturing polyacrylamide gels

a)     Run in 6% polyacrylamide gel with 7.5 M urea.

b)     Prepare the samples as follows:

  Mix 5 ml of amplified DNA with 3 ml of loading buffer for PAGE.

c)     Denature the samples for 5 min in thermocycler.

        Load gel extremes with 1 kb and 50 bp markers (10 ml. )

d )    Place in the electrophoresis chamber:

        "upper buffer"                 TBE   0.5X

        "lower buffer”                  TBE     1X  

5.     For gel preparation and staining, follow corresponding protocols