Guidelines for setting up an Antisense nested PCR

These guidelines relate to a protocol which uses an annealing temperature of 72°C for a high-temperature phase of 15 cycles and 58°C for a subsequent low-temperature phase. However the guidelines are flexible and the parameters can be readily adjusted for different protocols.

When using antisense PCR to quantify rare targets, when the PCR typically gives Ct values of >35, it may be useful to use 20 cycles during the high-temperature phase and possibly to use a higher annealing temperature during the low-temperature phase.

Note that the PCR protocol uses an annealing time of 30 sec for the high-temperature phase followed by 5 min incubation at 58°C, and 90 sec annealing times for the low-temperature phase. We have less experience at using shorter times for the low-temperature phase, but if these are used then our results suggest that the antisense effect may need to be increased by, for example, using a higher Tm oligonucleotide or doubling the oligonucleotide concentration. If an unwanted antisense effect is observed during the high-temperature phase then decrease the annealing time during this phase.

Outer primers

Design for a Tm of 74°C.

Test over a gradient of annealing temp of 68–74°C to ensure primers work well at 72°C. Do 20 cycles at the gradient temperature and then the remaining cycles at 58°C to ensure that the probe is active.

Also, as a control, synthesise the (extended) primer with the 3′ extension that would be produced by the antisense oligo. Test that the extended outer primers do not inhibit the activity of the inner primers in a PCR. The efficacy of the extension in preventing amplification can be tested by comparing the extended primer to the native primer.

Inner primers

Design for a Tm of 59–62. Ensure that there are no homologies, particularly at the 3′ end, between the inner primers and (i) the native or extended outer primers, and (ii) the antisense oligonucleotides

Synthesize 2 pairs, to bracket Tm range.

Test over a gradient of annealing temperature of 56–65 to ensure that Ct is maintained at temperature <= 60 but starts to lengthen above this temperature.

Antisense oligonucleotides

Design the core sequence, excluding tags, to a Tm of 50–55. Suggest synthesise 2 pairs within this range. Our current recommendations differ in several respects from the original protocol described by Brisco and Morley.

• The original design used, on grounds of cost, a 3′ tag comprising 3 bases which were the same as the 3 bases of the primer to which the tag was apposed. We now recommend the synthesis of oligos chemically blocked, e.g. amino blocked, at the 3′ end, at least for the antisense oligonucleotides which are finally used. Blocked oligonucleotides will be unable to produce nonspecific amplification from targets elsewhere in the genome. If desired on grounds of cost, initial exploratory studies can use oligonucleotides with 2 or 3 base mismatches at the 3′ end. However, if this is done, if possible avoid using G or C in order to avoid mis-priming off targets elsewhere in the genome.
• We now recommend that the 5′ Tag has only 2 bases, as this seems to produce a more powerful antisense effect. In addition, if possible use A or T, rather than G or C, so that the extended primer will have T or A at its 3′ end and will have less chance of mis-priming from elsewhere in the genome. This design feature now seems to us more important than having a sequence complementary to the 2 bases of the template immediately 5′ (in terms of the template strand) of the ultimate 5′ base to which the native primer binds.
• Test each oligo by setting up a PCR using 15 cycles at 72°C, subsequent cycles at 58°C and amounts of DNA which will lead to Ct results of approximately 23 and 28 (e.g. 50 ng and 2 ng). First, compare each outer primer plus its antisense to the outer primer alone. With 50 ng, antisense should increase the Ct value by 1-3 cycles, and, with 2 ng, antisense should increase Ct by >5 cycles. Second, having chosen a candidate pair of oligos, test the pair. With 50 ng, antisenses should increase the Ct value by 5–8 cycles, and, with 2 ng, antisenses should result in a very prolonged Ct or no detectable value.

It is often easier to obtain the desired to effect of the antisense oligonucleotide by varying its concentration.

To ensure that antisense pair does not cause measurable inhibition during the high-temperature phase, compare outer primers plus antisense pair versus outer primers alone, run the PCR entirely at the conditions for the first phase, and use 50, 5 and 0.5 ng DNA to provide Ct values approximately 23–30. Usually there is no measurable inhibition until the Ct becomes >=30. Any antisense effect during the high-temperature phase can be decreased by decreasing the annealing time during this phase to 15 or even 5 secs.

Complete System

Our experience is that these endpoints for testing of the primers and antisense oligonucleotides are simple to achieve and enable the complete system to be established.

The complete system can be tested over a range of input DNA, from 50 ng to 50 pg, in a comparison with the outer primers only. Ct values should be essentially the same over this range.

As a control test, select an unrelated PCR which is active when the 2 phase temperature protocol is used, and test that adding the extended outer primers together with the antisense pair does not result in inhibition.