PCR for Testing for GM Food

 

Credits:  Susie Stevens of Latta High School, Oklahoma; reported protocol derived from DNA Learning Center of Cold Spring Harbor Laboratory.

 

For a discussion of the purpose of Bt corn, go to http://www.extension.umn.edu/distribution/cropsystems/DC7055.html  

 

Another location http://croptechnology.unl.edu/download.cgi has animations of many plant-related concepts, including ECB and Bt – an animation of how Bt causes death in the European corn borer (click on diagram to activate) and further down the list, under Ps, an animation of how PCR, polymerase chain reaction, works.

 

Part A. Isolation of DNA from Food Samples or Plants:  Method One

Materials and Equipment:

PrepManTM Ultra Sample Preparation Buffer (#4322547 from Applied Biosystems)

Sterile micropestles

Boiling water bath

Microcentrifuge

Isopropanol

Distilled water

3M potassium acetate

Micropipettor with tips

Microtubes (1.5 mL)

TE Buffer

Corn food samples or corn plant leaves, Bt+ or Bt-

 

Procedure:

  1. Purchase food samples or obtain plant samples and assign.
  2. Pulverize food samples that are hard or crunchy.
  3. Place very small amount of food sample in 1.5 mL microtube (<100 ml volume).
  4. Add 400 ml of PrepMan Ultra Buffer reagent and use pestle to grind food for one minute.
  5. Vortex 10 seconds.
  6. Put in boiling water bath for 10 minutes; cool 2 minutes at room temperature.
  7. Centrifuge 3 minutes at 16,000 x g.
  8. Transfer 75 ml of supernatant into clean microtube and add 75 ml of distilled water.  (Okay to stop here, freeze samples and resume procedure in later class period.)
  9. Add 50 ml of 3M potassium acetate (to precipitate proteins).
  10. Add 155 ml of isopropanol, mix, leave at room temperature for 3 minutes.
  11. Centrifuge at 14,000 x g for 6 minutes; decant supernatant.
  12. Centrifuge again as above and pipette out any remaining alcohol.
  13. Let pellet air dry for 10 minutes.
  14. Resuspend DNA pellet in 100 ml of TE buffer.
  15. STOP POINT.  Store DNA template at -20o C.

 

Part A. Isolation of DNA from Food Samples or Plants:  Method Two

 

Materials and Equipment

Saline solution (0.9%NaCl), 1 mL

10% Chelex, 100 ml

Sterile micropestles

Boiling water bath

Cup of ice

Microcentrifuge

Distilled water

Micropipettor with tips

Microtubes (1.5 mL)

TE Buffer

 

Procedure

  1. Purchase food samples or obtain plant samples (1 cm x 1 cm) and assign.
  2. Pulverize food samples that are hard or crunchy.
  3. Place very small amount of food sample in 1.5 mL microtube (<100 ml volume).
  4. Add 1 mL of saline solution and use pestle to grind food or plant for one minute.
  5. Centrifuge 1 minute at highest speed of microcentrifuge (using balanced configuration of tubes).
  6. Carefully pour off supernatant and discard.  Be careful not to disturb cell pellet at bottom of test tube.  A small amount of saline can remain in the tube.
  7. Resuspend pellet by pipetting in and out (add up to 30 ml saline if needed).
  8. Withdraw 30 ml of cell suspension, and add to tube containing 100 ml of chelex.  Shake well to mix.
  9. Put in boiling water bath for 10 minutes; cool 2 minutes on ice.
  10. Shake tube.  Centrifuge 1 minute at 16,000 x g.
  11. Transfer 60 ml of supernatant into clean microtube.  Avoid cell debris and chelex beads.  This is your DNA sample.  (Okay to stop here, freeze samples and resume procedure in later class period.)
  12. STOP POINT.  Store DNA template at -20o C. or on ice till ready to begin part II.

 

II.                Part B.  Amplification of 35S promoter (CaMV) (161 bps) and Bt Cry1A gene (146 bp) by PCR

Materials and Equipment

Thermal cycler

micropipettors and sterile aerosol-barrier tips

Ready-to-go PCR Beads (Amersham Biosciences #27-9558)

Microtubes

Deionized distilled water

Template DNA isolated in Part A from food or plant samples

*Cresol red loading dye:  Add 17 g. sucrose to 49 mL of sterile distilled water; dissolve.  Add 1 mL of 1% cresol red dye solution.  Mix. Store at 4o C.  Makes 50 mL.

35S primer mix:

            515 ml of distilled water

            460 ml of cresol red loading dye*

75 ml of 5’ and 3’ primers at 15 pmols/ml using following primer sequences:

Forward 5’ CCG ACA GTG GTC CCA AAG ATG GAC

Reverse 5’ ATA TAG AGG AAG GGT CTT GCG AAG G

 

Bt primer mix:

            515 ml of distilled water

            460 ml of cresol red loading dye

            75 ml of 5’ and 3’ primers at 15 pmols/ml using the following primer sequences:

            Forward 5’ CTG GTG GAC ATC ATC TGG GGC ATC TTC G

            Reverse 5’ TTG GTA CAG GTT GCT CAG GCC CTC C

(Suggestion:  Heat primers one minute at boiling or near-boiling before use from freezer storage)

Procedure:

  1. Each sample would have two PCR reaction tubes, each containing PCR ready-to-go bead:  1 for 35S primer and 1 for Bt primer.  Label tubes.
  2. Add 22.5 ml of Bt primer mix to one tube, 35S primer mix to other tube.  Tap tube to dissolve bead completely in mix.
  3. Use fresh tip to add 2.5 ml of sample DNA to each reaction tube and mix completely.  Pool reagents by quick spin in microcentrifuge or by sharply tapping the tube bottom on the lab table.
  4. Store on ice till ready to put into thermal cycler; move into thermal cycler as quickly as possible.
  5. Use the following program:

15 cycles of: 98oC for 20 sec, 58oC for 4 sec, 72oC for 15 sec

15 cycles of: 98oC for 20 sec, 54oC for 4 sec, 72oC for 15 sec

15 cycles of: 98oC for 20 sec, 50oC for 4 sec, 72oC for 15 sec

Hold: 4oC

 STOP POINT. Store PCR product tubes in -20 degree freezer till ready to run gels.

Part C.  Gel Analysis of PCR Results

Materials:

2% agarose gel made in TBE

TBE buffer

PCR samples from part B

1 mg/mL ethidium bromide

micropipettors and tips

electrophoresis chamber and power supply

gloves

100 bp DNA marker (ladder)

UV transilluminator

 

Procedure:

 

  1. Load 15 ml of each PCR sample into wells of 2% agarose gel.
  2. Load 10 ml of 100 bp DNA marker into another well.
  3. Electrophorese at 130 volts for 20-30 minutes.
  4. Stain gel in an ethidium bromide solution (1 mg/mL) for 10-15 minutes.
  5. Observe gel under UV to detect bands.  If you are using a 100 bp Ladder, the Bt gene (cryA1) (146 bp) and the 35S promoter gene (161 bp) should appear as a band between the 100 bp and 200 bp fragments of Ladder.  Smeary bands below 100 bps in size usually represent primer-dimers.

 

Results: