Mesoplasma florum:Inverse PCR Transposon location

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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.


Contents

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

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