Pit-stop PCR: an approach to increase final product yield of multiplex PCR.

Since the development of the polymerase chain reaction (PCR), many strategies have been designed to amplify one or more specific DNA sequences with a single set of oligonucleotide primers. Multiplex PCR is a strategy to amplify simultaneously either two or multiple DNA targets sequences in a single reaction (4). A variety of reaction conditions must be used to optimize multiplex-PCR strategies. However, increasing the number of primer pairs in a single PCR increases the chance of obtaining spurious amplification products. In a number of cases PCR primer pairs do not maintain their specificity when combined into a single reaction mixture using the amplification conditions established for the individual pairs (3), and some PCR products are produced at greater efficiencies than others. An approach to solving this problem is to change primer concentrations, which could prove to be insufficient. In this report, a simple and efficient modification is described to improve the signal in three different experimental conditions of multiplex PCR. We propose naming this strategy pit-stop PCR. A number of multiplexing protocols, e.g., for bovine embryo sexing by PCR using bovine Y-chromosome-specific primers and the co-amplification of bovine autosomal primers as an internal control for specimen integrity have been described (8,9). When using bovine autosomal control primers in the described reaction, the presence of two bands (one male-specific and one autosomal bovine-specific) is interpreted to be a male embryo; whereas, when the male-specific band is absent and the bovine-specific band visible, the embryo is considered to be a female. The lack of the two bands indicates the absence of embryonic DNA in the sample, which is considered a dubious diagnosis. Thus, using this control, sex misidentification did not occur (excluding the false female results). However, when no autosomal control primers are used, the sample showing one amplified band is considered as a male, and when no male-specific band is detected, the embryo is considered to be a female one. In this case, the presence of no amplified products could also indicate the absence of embryonic DNA in the sample. In spite of the use of a control primer pair being helpful for avoiding the occurrence of false sex identification, many autosomal oligonucleotides tested seem to interfere with the amplification of the Y-chromosome-specific sequence, especially when a very dilute amount of template was used (2,8). In this work, a multiplex-PCR protocol for sex determination of bovine embryos was tested. The pair of bovine Ychromosome-specific oligonucleotides (6) was designed from a bovine repetitive sequence of Y chromosome to amplify a 175-bp product. The pair of control bovine-specific oligonucleotide primers (8) was designed to amplify a 216-bp fragment of a 1399-bp-long repeated unit of bovine 1.715 satellite DNA. The male and female bovine DNAs used were isolated from heparinized peripheral blood (7) or from embryonic cells (5). The PCR was performed in 0.5-mL microcentrifuge tubes containing a final concentration of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 100 μM of each dNTP (Boehringer Mannheim GmbH, Mannheim, Germany), 0.4 μg bovine serum albumin (BSA) (nuclease-free; Cenbiot, Porto Alegre, RS, Brazil), 0.6 ng each of oligonucleotide primer (Biodynamics, Buenos Aires, Argentina), 1 U per assay of Taq DNA Polymerase (Cenbiot) and male or female bovine DNA as template. Mineral oil (40–50 μL; Sigma, St. Louis, MO, USA) was pipetted on top of the reaction mixture (25 μL) and incubated at 94°C for 5 min in a DNA Thermal Cycler (PE Biosystems, Foster City, CA, USA). The PCR program consisted of 40 cycles at 94°C for 60 s, 56°C for 30 s and 72°C for 60 s, followed by a final extension at 72°C for 8 min. The PCR products were analyzed on agarose gels. Optimization involved varying enzyme concentration, buffer composition and cycle parameters of the multiplex-PCR protocol. Nevertheless, some PCR products were produced at greater efficiency than others, which is consistent with a very weak or null amplification of the Y-specific fragment commonly observed in bovine DNA male samples. To solve this problem, either the primer concentrations were reduced for the PCR products that produced the greatest yields or the primer concentrations were increased for PCR products with the lowest yields. However, these latter approaches resulted in no improvement in signal of the lowestyielding PCR product. A modification was then introduced in the multiplex-PCR strategy that consisted of including the pair of bovinespecific control primers, which produced the greatest amount of PCR product, into tubes containing the reaction mixture only after several cycles. The best results of this pit-stop PCR strategy were obtained when the pair of bovine-specific control primers was added after the fourth cycle of the DNA amplification (Figure 1). This pit-stop PCR strategy was also used to solve similar problems with two other distinct multiplex PCRs as described elsewhere (1,10). The weaker signals produced by (i) the femA gene PCR product after the co-amplification