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,
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.
religating, and using outward directed primers from the transposon insertion to amplify across the religation junction.

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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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