Introduction

Many PCR techniques have been used to genotype apolipoprotein E (APOE). These include real-time PCR (1), capillary electrophoresis with laser-induced fluorescence (2), denaturing high-performance liquid chromatography (3), TaqMan® assays (4), matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and homogeneous mass-extend technology (5), allele-specific PCR (6), and real-time PCR combined with fluorescence resonance energy transfer hybridization probes (7).

Isolated-probe PCR (IP-PCR) is a new technique that separates unlabeled probes from asymmetric PCR. The unlabeled probes are placed in the top of the capillary tubes. This prevents the probes from interfering with the asymmetric amplification step. After amplification, the LightCycler® capillary tubes are inverted and centrifuged using a desktop centrifuge, forcing the PCR mixture into the cap to mix the unlabeled probes and the PCR mixture. An additional denaturing and annealing cycle is needed for the probes to hybridize to the template to create a detectable signal. The samples are then melted using a high-resolution melting instrument to determine the six different APOE genotypes.

Asymmetric PCR selectively amplifies one strand of the genomic DNA (8). This is accomplished by using unequal concentrations of forward and reverse primers. The optimum asymmetric ratio of forward to reverse primers needs to be determined experimentally for each target (9). The PCR mixture contains a double-stranded dye LCGreen® I (Idaho Technology, Salt Lake City, UT, USA) that fluoresces when it is bound to double-stranded DNA (dsDNA) (10).

In conventional asymmetric PCR, the probes need to have a melting temperature lower than the extension temperature so that they do not hybridize to the DNA template when the polymerase is extending the primers (9). This limits the probe design and makes it difficult to design probes that are long enough to produce a strong fluorescent signal, especially in high-GC targets like APOE. Primer and probe design for asymmetric PCR often requires several attempts to obtain a set of compatible primers and unlabeled probes that will amplify the specific target and produce an adequate signal.

Optimization of IP-PCR is straightforward because there are no probes in the PCR to interfere with the primers or the DNA polymerase. Designing probes for IP-PCR is also straight-forward; the only restriction being that the probe melting temperature must be distinguishable from the PCR product peak. IP-PCR also allows multiple unlabeled probes to be multiplexed in one reaction as long as they have different melting temperatures.

Materials and Methods

DNA Isolation

Previously genotyped whole blood samples of the six APOE genotypes were obtained from the molecular pathology laboratory at ARUP (Salt Lake City, UT, USA). These samples were genotyped at ARUP using a LightCycler ApoE Mutation Detection kit (Roche Applied Science, Indianapolis, IN, USA). The samples were then deidentified for use in this experiment. The DNA was isolated using the PUREGENE® Genomic DNA Purification kit from Gentra Systems (Minneapolis, MN, USA).

Primer and Probe Design

Both the forward and reverse primers were designed using a DNA melting temperature calculator program that was written using LabVIEW (National Instruments, Austin, TX, USA). The sequence of the forward primer is 5′-ACGCGGGCACGGCTGTCCAAGG-3′. The reverse primer sequence is 5′-GGCGCTCGCGGATGGCGCTGA-3′. The forward and reverse primers amplify a PCR product 277-base pairs-long with a melting temperature of 93.1°C and a GC content of 75%. The GenBank® accession no. for APOE is K00396.

The APOE 112 probe sequence is 5′-ACATGGAGGACGTGTGCGGCCGCCTG-P-3′. The APOE 158 probe sequence is 5′-GCGGCTCCTCCGCGATGCCGATGACCTGCAGAAGCGCCTGGC-P-3′. The SNP position is indicated by the underlined and bold text.

All primers and probes were synthesized by the Core Facility at the University of Utah (Salt Lake City, UT, USA).

Asymmetric PCR with Isolated Probes

PCR was performed in 10-µ/mL reaction volumes with 50 mM Tris buffer, pH 8.3, 500 µg/mL bovine serum albumin (BSA), 2 mM MgCl₂, 200 µM each deoxynucleotide triphosphate, 0.4 U KlenTaq1™ polymerase (Ab Peptides, St. Louis, MO, USA), 1× LCGreen I, 7% dimethyl sulfoxide (DMSO), 0.05 µM forward primer, 0.5 µM reverse primer, and 5 ng/µL human DNA. This mixture was pipeted into the capillary tube. It was next centrifuged down to the bottom of the capillary tube using a desktop centrifuge at 2000× g for 3–4 s. Then, 2 µL probe mixture containing 0.25 µM both 112 and 158 probes were pipeted into the top plastic well of the capillary tube. Finally, the capillary tube was capped.