Collaborative Instructional Project:  Guatemala-Molecular Genetics Lab: Jan. 00 to March 00

Mr. Steve Hong, a graduate student, is doing the lab work at UW-Madison in Douglas P. Maxwell's Lab.

Updated Feb. 2, 2000 by DPM.

Objective:  Molecular characterization of the geminivirus (es) associated with tomato samples collected in Sansirisay, GT January 11, 2000

Samples: (tissue cut into small strips and air dried)

   i)  Mandarina-1, DSI = 2, commercial plot, west side of Rio los Platanos

   ii)  Elios-1, DSI = 4, germplasm evaluation plot

Approach:

   i)  DNA extraction by the Dellaporta method (see Rojas et al., 1993)

   ii)  PCR with top A primers (PAL1v1978/PAR1c715, Rojas et al., 1993) and coat protein primers (514/1048, Wyatt and Brown, 1996)

   iii)  PCR amplified fragments cloned into a T-Easy vector (3,015 bp, pGEM-T Easy vector) from Promega Corp. (www.promega.com), this vector has blue/white selection, also T overhangs for easy of cloning PCR fragments.

   iv)  Transformation of recombinant plasmid into XL-1 Blue strain of E. coli and plating of cells on 2x YT agar medium with amp and tet added, plus x-gal and IPTG.

   v)  White colonies saved on another agar plate, and also, transferred to  liquid 2x YT medium with amp and tet and grown over night at 37 C.  Miniprep DNA prepared from each of these cultures.

   vi)  Miniprep DNA digested with EcoRI endonuclase since there is an EcoRI site on each side of the cloning site so that a fragment of the size of your PCR fragment will be obtained, if there are no internal EcoRI sites in your PCR fragment.

   vii)  Once a recombinant plasmid is obtained with the correct size insert, more recombinant plasmid DNA is obtained and this DNA is taken to the Biotechnology Center where it will be sequenced using the dideoxynucleotide chain termination method with each dideoxynucleotide labeled with a different color fluorescing dye, which is detect by a spectrophotometer.

   viii)  Sequence will be available from both ends of the insert since two sequencing primers are used.  These sequencing primers anneal 5' of each of the EcoRI sites on either the + or the - DNA strand.

   ix)  Sequence can be analyzed for comparison to other DNA sequences at the GenBank database with the internet.

References:

Rojas, M. R., Gilbertson, R. L., Russell, D. R., and Maxwell, D. P.  1993.  Use of degenerate primers in the polymerase chain reaction to detect whitefly-transmitted geminiviruses.  Plant Dis. 77:340-347.

Wyatt, S. D., and Brown, J. K.  1996.  Detection of subgroup III geminivirus isolates in leaf extracts by degenerate primers and polymerase chain reaction.  Phytopathology 86:1288-1293.

[International Plant Virology Lab]