SINGLE-FLY DNA PREPS FOR PCR

SINGLE-FLY DNA PREPS FOR PCR

Gloor et al. 1993 Genetics 135:81-95

We have developed a simple method for the rapid and reproducible isolation of DNA from single flies for amplification by the polymerase chain reaction (PCR) (Saiki et al, Science 239: 487), and direct sequencing by asymmetric PCR (Gyllensten and Erlich, Proc. Nat. Acad. Sci. 85: 7652). The simplicity of this procedure means that the problem of contamination with other amplified or cloned DNA is greatly reduced. Sufficient DNA is obtained from one fly for a minimum of 50 PCR analyses, and the DNA is stable for at least one month in the refrigerator. A simple modification of this technique allows the isolation of DNA suitable for use in inverse PCR (Ochman et al, Genetics 120: 621-623). These methods substantially reduce the time involved in DNA isolation, and among other uses, allows the PCR to be used to monitor the segregation of an allele for which there is no phenotype or transposition of an unmarked P element (Engels et al. Cell 62: 515-525).

A. DNA PREPARATION PROTOCOL:

  1. The squishing buffer (SB) is 10 mM Tris-Cl pH 8.2, 1 mM EDTA, 25 mM NaCl, and 200 ug/ml Proteinase K, with the enzyme diluted fresh from a frozen stock each day.

  2. Place one fly in a 0.5 ml tube and mash the fly for 5 - 10 seconds with a pipette tip containing 50 ul of SB, without expelling any liquid (sufficient liquid escapes from the tip). Then expel the remaining SB.

  3. Incubate at 25-37 C (or room temp.) for 20-30 minutes.

  4. Inactivate the Proteinase K by heating to 95 C for 1-2 minutes.

    NOTES: This preparation can be stored at 4 C for months. We typically use 1 ul of the DNA prep in a 10-15 ul reaction volume. It does not matter if fly parts (wings, bristles, legs) are inadvertantly added to the PCR mixture. Product will typically start to appear after 24-25 cycles, but 28-30 cycles seems to give maximal yield. Increasing the number of flies does not seem to increase the signal significantly, probably due to increasing concentrations of inhibitors. There should be no problem scaling up the number of flies screened if the volume is increased proportionately. 85 C heat inactivation allows longer fragments to be amplified -- up to more than 9 kb with ³long PCR²

B. DNA PREPS FROM MANY INDIVIDUAL FLIES

A similar method can be used with 96-well (8 x 12) micro plates to prepare DNA from a large number of individual flies. Up to 80 flies can be tested with a single plate.

  1. Place one anesthetized or frozen fly in each well. All rows (A-H) can be utilized, but leave the first and last columns (1 and 12) empty to ensure complete heating in step 3.

  2. Add 50 ul of SB to each well and macerate each fly with a toothpick for 5-10 seconds. Then cover the plate with an adhesive-backed strip to prevent evaporation and contamination. Incubate at room temperature as before.

  3. Use two standard-size heating blocks (9.5 x 7.5 cm) pre-heated to 95 C to inactivate the Proteinase K. Sandwich the micro plate, with its adhesive lid still in place, between the two heating blocks. The lower block should be inverted so that the tube holes are facing downward and the flat surface is touching the bottom of the micro plate. After 2-3 minutes remove the micro plate and set it on the bench top with the upper hot block still on top. This upper block will gradually cool to room temperature, preventing condensation on the underside of the adhesive.

    NOTES: It is helpful to tape a piece of waxed paper over the open micro plate while the PCR tubes are being set up. That way the DNA samples can be drawn from each well by poking the pipet tip through the waxed paper. This procedure reduces the possibility of contamination and helps to keep track of which wells have been used.

C. INVERSE PCR

PMSF (phenylmethylsulfonylfluoride) can be used instead of the 95 C treatment to inactivate the Proteinase K if the DNA preps are to be used for inverse PCR or other methods that require double-stranded DNA.

  1. Add 1 ul of 0.1 M PMSF to the fly prep following step 3 of protocol A above. Then heat the mixture to 65 C for 10 - 15 minutes to denature any proteins not inactivated by the proteinase.

  2. Add 4 ul of fly supernatant to 16 ul of 1.25X NdeII buffer (125 mM Tris-HCl pH 7.6, 12.5 mM MgCl[2], 188 mM NaCl, 1.25 mM DTT). Add 0.5 ul of NdeII (BRL) and incubate at room temp. for 15 min. Inactivate the enzyme by heating to 65 C for 15 min.

  3. Take 3 ul of digested DNA and add to 7 ul of ligation mix (5 mM MgCl2, 20 mM DTT, 0.8 mM ATP). Add 0.5 ul T4 DNA ligase (NEB) and incubate at room temperature for 20-30 min. Inactivate the enzyme by heating to 95 C for 2-3 min; this also serves to nick the DNA.

    NOTES: For the first attempt with a new insertion, we recommend using the following conditions: denature at 94 C for 45 sec., anneal at 60 C for 45 sec., extend at 72 C for 4 min; try 30 - 35 cycles. - The restriction enzyme appears to be the most critical component in this protocol. The enzyme must be specific under conditions of very low DNA concentration. Sau3A1, for example, is too promiscuous and digests at several sites in addition to its canonical restriction sequence. The protocol should work with other enzymes. The protocol has been used for a combined inverse/asymmetric PCR procedure to get DNA sequences flanking P element insertions.

Greg Gloor and William Engels, Dept. of Genetics, Univ. of Wisconsin, Madison, WI 53706, 608-263-2213, FAX/262-2976, WRENGELS@facstaff.wisc.edu