Vectorette PCR of mTn-3xHA transposons

(Text modified and expanded from that found at C. Friddle's web site.)

Identifying the insertion site by vectorette PCR

An adaptation of the method of Riley et al (1990) can be used to recover genomic DNA adjacent to the mTn insertion. Genomic DNA from the strain of interest is digested with a restriction enzyme to produce small, blunt-ended fragments. An 'anchor bubble' is ligated to the fragment ends. PCR is then performed using a primer complementary to mTn sequences, and a primer that is identical to sequence in the 'bubble' region (the universal vectorette or UV primer). In the first cycle of PCR, only the mTn primer can bind the template. In subsequent cycles, the UV primer can bind the product of the mTn primer. Thus, only the fragment containing the mTn primer binding site is amplified. The PCR product may then be isolated and sequenced. The vectorette PCR method has the advantage of eliminating transformation and cloning steps. It is also the only method currently available to determine the insertion site in a yeast genome mutagenized with mTns that do not contain lacZ.
  1. Prepare good quality genomic DNA (Philippsen et al., 1991, Burns et al; 1996) from the yeast strain carrying the transposon insertion. Cut 1-3 ug of DNA overnight with 8-10 u of AluI, in a total volume of 20 ul. Other frequent-cutting enzymes that leave blunt ends may be used, provided that there is at least one site for them in the mTn (to ensure that both terminal inverted repeats are not present on the fragment) and that this site does not lie between the mTn end and the binding site for the suggested mTn primer (see below).
  2. Heat inactivate the enzyme by heating digest to 65C for 20 min.
  3. Add 3 ul of 10x buffer used in restriction digest, 1 ul of annealed Anchor Bubble (see below), 1 ul of ligase (400 u), 0.5 ul of 5mM ATP and 24.5 ul of water. Incubate at 16oC for 9-24 hr.
  4. Set up a 100 ul PCR reaction with the following components: 5 ul of the ligation reaction, 2.5 ul of 20 uM mTn internal primer (see below), 2.5 ul of 20uM UV primer (see below), 8 ul of 2.5 mM dNTPs, 10 ul of 10x Taq PCR buffer, 71 ul water and 1 ul Taq DNA polymerase (5 u). A 'hot start' using Ampliwax (Perkin Elmer) is recommended.
  5. Transfer to thermal cycler and denature at 92C for 2 min. Perform 35 cycles of 20 sec. at 92C, 30 sec. at 67C and 45-180 sec. at 72C (use the longer interval if a product is not apparent), followed by a single cycle of 90 sec. at 72C.
  6. Run 80 ul of the PCR on a 1-3% SeaKem GTG agarose (FMC) gel. Usually only 1 band containing 200-400 ng of DNA is seen. Excise all bands individually and recover DNA with Qiaex (Qiagen). Elute DNA with 12 ul of TE.
  7. Sequence 7 ul of recovered DNA with Sequenase kit (Amersham). Use 200-600 pMols (yes, really) of M13(-47) primer (or GFP primer for mTn-3xHA/GFP), and high specific activity (>1000 Ci/mmol) S35 labelled nucleotide (Amersham). Denature sequencing reactions by boiling for 10 min., followed by imediate cooling in ice water.

Vectorette primers

Possible mTn internal primers

Accession #s

References