Mesoplasma florum:Inverse PCR Transposon location

We identify the location of the transposon insertion event by cutting the genomic DNA with a frequent cutter, religating, and using outward directed primers from the transposon insertion to amplify across the religation junction. Sequencing the resulting fragment identifies the insertion location.

Choice of Enzyme

 * Hutchison used DraI as a cutter, but this is a blunt cutter, making religation difficult
 * MboI cuts at GATC sites, and is insensitive to 5-me dCTP methylation (sensitive to methylation of A)
 * MboI cutting frequency calculation: p(cut) = (.13)(.37)(.37)(.13) = .00231
 * Expected fragment length = 1/ .00231 = 432 bp + length from primer site to end of transposon
 * Expected PCR fragment length is twice this length, or about 1Kbp

Materials

 * Genomic DNA from single colony transposon insertion event
 * MboI
 * NEB buffer 3 10x
 * T4 DNA ligase buffer 10x
 * T4 DNA ligase
 * PCR supermix
 * M13forward(-47) primer
 * T7 universal primer
 * ME primer
 * E-Gel 0.8%

Restriction digest of 500 ng of genomic DNA with MboI

 * Mix 0.5 &mu;l (approx) genomic DNA in TE
 * 5 &mu;l NEB buffer 3
 * 1 &mu;l MboI
 * 43.5 &mu;l water
 * incubate at 37&deg; for 30 minutes
 * heat kill at 65&deg; for 20 minutes

Ligation of cut ends at 5 ng/&mu;l concentration (Hutchison99)

 * 5 &mu;l digested DNA
 * 1 &mu;l T4 DNA ligase buffer
 * 0.2 &mu;l T4 DNA ligase
 * 3.8 &mu;l water
 * incubate 10 minutes at room temperature

Transposon detection PCR reaction 10 &mu;l test volume
9.2 &mu;l PCR supermix High Fidelity
 * 0.5 &mu;l ligated DNA
 * 0.3 &mu;l ME primer

Inverse PCR for transposon location identification

 * 0.5 &mu;l ligated DNA
 * 0.15 &mu;l M13forward(-47) primer
 * 0.15 &mu;l T7 universal primer
 * 9.2 &mu;l PCR supermix high fidelity


 * Cycle 5 minutes at 95&deg; initial denturation
 * 40 cycles of
 * 94&deg; 30 seconds
 * 55&deg; 30 seconds
 * 64&deg; 3 minutes
 * 10 minutes 64&deg; final extension

Detect with E-Gel 0.8%

Inverse PCR for sequencing transposon location

 * 5 &mu;l ligated DNA
 * 1.5 &mu;l M13forward(-47) primer
 * 1.5 &mu;l T7 universal primer
 * 92 &mu;l PCR supermix high fidelity
 * Cycle as above
 * Prepare 1% agarose prep gel
 * add 20 &mu;l of loading dye
 * Run gel
 * Cut the band
 * Prepare DNA the gel with Qiaex II kit
 * Quantitate DNA
 * Sequence with both M13forward(-47) primer and T7 universal primer

Sequence analysis

 * locate the MboI cut site (GATC) in the sequencing results
 * locate the end of the transposon sequence (ME end, reverse complemented here, agatgtgtataagagacag)
 * Identify the duplicated 9bp insertion site surrounding the insertion event
 * Locate the sequence from the ME end to the GATC cut site on the Mesoplasma florum genome
 * The sequence from the M13F(-47) and T7 Universal primers should be adjacent, and oriented in opposite directions on the genome