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 μl (approx) genomic DNA in TE
• 5 μl NEB buffer 3
• 1 μl MboI
• 43.5 μl water
• incubate at 37° for 30 minutes
• heat kill at 65° for 20 minutes

Ligation of cut ends at 5 ng/μl concentration (Hutchison99)

• 5 μl digested DNA
• 1 μl T4 DNA ligase buffer
• 0.2 μl T4 DNA ligase
• 3.8 μl water
• incubate 10 minutes at room temperature

Transposon detection PCR reaction 10 μl test volume

• 0.5 μl ligated DNA
• 0.3 μl ME primer

9.2 μl PCR supermix High Fidelity

Inverse PCR for transposon location identification

• 0.5 μl ligated DNA
• 0.15 μl M13forward(-47) primer
• 0.15 μl T7 universal primer
• 9.2 μl PCR supermix high fidelity

• Cycle 5 minutes at 95° initial denturation
• 40 cycles of
  • 94° 30 seconds
  • 55° 30 seconds
  • 64° 3 minutes
• 10 minutes 64° final extension

Detect with E-Gel 0.8%

Inverse PCR for sequencing transposon location

• 5 μl ligated DNA
• 1.5 μl M13forward(-47) primer
• 1.5 μl T7 universal primer
• 92 μl PCR supermix high fidelity
• Cycle as above
• Prepare 1% agarose prep gel
• add 20 μl 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

References

Hutchison, CA et. al, Global transposon mutagenesis and a minimal Mycoplasma genome. PMID 10591650