Rescuing Insertion to Identify Mutation

Once an interesting mutation is purified to homokaryonicity, identifying the gene that carries the insertion is initiated. An initial step in this regard is to determine how many insertions are found per mutant. This could be determined either by performing a sexual cross and following transmission of the trait using classical genetics, or more simply, with a Southern blot (described here).

1. Inoculate the conidia of each mutant line into liquid minimal medium. After achieving a tissue mass of about 1 cm3, blot the mycelia dry on paper towels and freeze in a -70°C freezer.

2. Grind the mycelia in liquid nitrogen using a mortar and pestle with the aid of a pinch of sand and suspend the powder in 0.5 mL of CTAB buffer.

3. Vortex and incubate at 60°C for 30 min.

4. Add 0.5 mL of C/IAA and extract the genomic DNA for 5 min with mixing.

5. Spin at 10,000g for 30 min at room temperature.

6. Collect the supernatant in a clean tube and repeat steps 4 and 5.

7. Add 1 ^L of RNase A (10 mg/mL) and incubate the mixture for 10 min at 37°C.

8. Precipitate the DNA with isoproponal at room temperature for 30 min. Collect the precipitate by centrifugation for 10 min at 10,000g, and wash it once with 70% ice-cold ethanol. After drying the pellet, the DNA can be suspended in 50 ^L water and quantitated by standard methods.

9. Digest 5 to 10 ^g of genomic DNA with £coRI, which does not cut within the pSKbar2 plasmid, thus yielding a single band per insertion.

10. Following digestion, separate the DNA on a 1% agarose gel, and blot according to standard methods.

Fig. 3. Southern blot analysis of insertions. Genomic DNA of 10 mutants was digested with EcoRI, which does not cut within the inserted plasmid sequence. Strains with single inserts have one band (first and last four lanes). The mutants in the middle two lanes carry two copies of the Basta-resistant gene. The arrow indicates a molecular weight of 4 kbp, the size of the plasmid alone.

Fig. 3. Southern blot analysis of insertions. Genomic DNA of 10 mutants was digested with EcoRI, which does not cut within the inserted plasmid sequence. Strains with single inserts have one band (first and last four lanes). The mutants in the middle two lanes carry two copies of the Basta-resistant gene. The arrow indicates a molecular weight of 4 kbp, the size of the plasmid alone.

11. Using primers bar2.1 and bar2.2 and the pKSbar2 plasmid as template DNA, amplify the Basta resistance cassette by PCR.

12. Label the PCR product by random priming using either radioactivity or digoxygenin, and then use it to probe the blot.

Insertional mutagenesis often yields a single insertion, but there can also be several (see Fig. 3). In the latter case, there may be multiple insertions spread throughout the genome or at a single site. Mutants with a single insertion are suitable for rescue. To identify the insertion, we used a combination method, starting with size selection of genomic fragments, ligation, and PCR. The fragments are ligated into a vector and the mixture is subjected to PCR using known sequences from the Basta resistance gene and within the vector (see Fig. 4 and Note 8).

1. Map the area of the insertion by standard methods (i.e., Southern blot), digesting the genomic DNA of the mutant with several restriction enzymes, which do not cut within the inserted plasmid, in conjunction with HindHI, which is known to cut within the Basta resistance gene (the minimal fragment that must be inserted for the selection). The primary goal is to identify a fragment that runs from the known HindIII site in the resistance gene (see Fig. 1) into the disrupted gene. A fragment with two distinct sticky ends (only one of which is HindIII) must be identified. Also, the size of the fragment should be big enough to deliver sequence information but small enough to ligate efficiently.

2. Gel-purify the restricted fragments of genomic DNA that run with the appropriate molecular weight (see Note 9) and ligate them overnight into a plasmid vector (i.e., pSK-II) using standard methods (see Note 10).

3. Use 1 ^L of the ligation reaction as the DNA template for a PCR reaction, which employs a primer for the Basta resistance gene on one end (bar2.1), and a primer

Fig. 4

for either T3 or T7 sites on the other. Thanks to the mapping work, the orientation of the fragment with respect to the vector is known, as well as the predicted size of the PCR product.

4. Utilize 0.5 ^L of the first PCR as a template for a second PCR reaction, using a primer nested in the Basta resistance gene (bar2.2). A major fragment of the expected size should be apparent. This can be purified and sequenced. The product can be compared to the known plasmid sequence, as well as to the mapped DNA.

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