Stratagenes custom molecular beacon synthesis assists in detecting SNPs quickly and easily
University of Colorado Health Sciences Center, Denver, Colorado
We used Stratagenes custom molecular beaconsff, to detect a single nucleotide polymorphism (SNP) in a region of chromosome 10q25.1 and showed clear discrimination among the different genotypes carried in family members. This molecular beacons application provides a fast and simple method that can be performed in 2 hours in a 96-well format and is especially suitable for high-throughout, high-quality genotyping of SNPs.
SNPs are the most frequently found DNA sequence variations in the human genome (about 1 per 350 bp frequency).1 The ability to efficiently genotype SNPs is increasingly seen as fundamental to identifying genetic factors associated with complex diseases. There are a number of available methods for SNP genotyping, ranging from traditional gel-based formats to detection with the TaqMan system2 and DNA microarrays3 that have offered the promise of high-throughput screening.
Molecular beacons are hairpin-shaped fluorescent hybridization probes that can be used to monitor the accumulation of specific product in a closed-tube in real time PCR. The homogeneous format eliminates the need for gel electrophoresis, reduces the time, effort, and risk of contamination involved with performing PCR analysis. Molecular beacons contain a fluorophore and a quencher moiety at opposite ends of an oligonucleotide. The ends of the oligonucleotide are designed to be complementary to each other and can form a stem structure, while the intervening loop is complementary to a sequence within the amplified product. When the unhybridized probe is in solu tion, it adopts a hairpin structure that brings the fluorphore and quencher sufficiently close to each other to allow efficient quenching of the fluorophore. If, however, the molecular beacon is bound to its complementary target, the fluorophore and quencher are far enough apart that the fluorophore cannot be quenched and the molecular beacon fluoresces.
Hybridization of molecular beacons with their complementary target is highly specific; therefore, we designed molecular beacons to study one of the SNPs of our interest. Based on linkage and association studies in a large Arab family with 20 affected relatives, a diabetes susceptibility locus (IDDM17) maps to 10q25.1.4 IDDM 17 and its flanking markers are localized to a 120-kb region within a completely sequenced bacterial artificial chromosome (BAC) based on association studies of a number of SNPs. We genotyped a group of people in the family and determined the presence of three genotypes. The genotypes were complementary with probe-target homozygote (A/A), mismatched probe-target homozygote (G/G), and heterozygote (A/G) for the SNP.
The fluorescence of the molecular beacon was monitored in real time while the PCR reaction was taking place. The fluorescent signal was monitored and reported during each anneal-extend step when the molecular beacon was bound to its complementary target. Results were displayed on an amplification plot, which reflected the change in fluorescence during cycling. Figure 1 shows the dissociation curves of hybrids between molecular beacons and A/A homozygote (green dots) or G/G homozygote (black circles). Fluorescence was plotted as the derivative value on the Y-axis. In the temperature interval from 62C to 72C, the perfectly complementary probe-target hybrid elicited strong fluorescence, whereas the mismatched probe-target hybrid had significantly lower fluorescence; hence, a clear discrimination was seen between the targets containing a single nucleotide difference.
Figure 2 illustrates the results in a plot of threshold cycle (Ct) values where molecular beacons were used to analyze six samples and one no-template control (each sample was assayed in triplicate). Ct is defined as the cycle at which fluorescence is determined to be above background while the reaction is in the exponential phase. Samples that showed no detectable increase in fluorescence throughout the reaction (Ct=40) were considered negative. Two individuals (A/A homozygous on SNP11 shown as black closed diamonds) produced detectable fluorescence beginning at about cycle 29 (average Ct standard deviation=29.10.17) with molecular beacon SNP11A but showed no detectable fluorescence above the background (Ct=40) with SNP11G. On the other hand, two individuals (G/G homozygous on SNP11 shown as black open diamonds) had no increased fluorescence (Ct=40) with molecular beacon SNP11A, and both showed detectable fluorescence above cycle 28 (average Ct STD =28.50.30) with molecular beacon SNP11G. For the two heterozygous individuals (A/G heterozygous on SNP11 shown as green solid stars), similar Cts were seen with both molecular beacons: one had Ct values at close to 35 (average Ct STD=34.90.48), and another had Ct values at around 31 for both molecular beacons (average Ct STD=31.00.44). The different Ct values observed for the two heterozygous individuals were likely caused by a difference in DNA concentration between the samples.
From the distribution of data, the three genotypes were easily distinguished from one another with either of the two molecular beacons. The advantage of having both molecular beacons is to clearly differentiate a homozygote from a heterozygote. For example, one heterozygote had Cts of approximately 31 with both molecular beacons, and the Ct values were not dramatically different from the complementary homozygous targets A/A of 29 Ct with probe A or G/G of 28 Ct with probe G. The perfectly matched homozygotes (A/A or G/G) became mismatched with the other probe (G or A), which resulted in a Ct of 40, so it was easily distinguished from heterozygous target (Ct=31). Negative control samples (shown as black closed dots) containing no template also generated no fluorescence (Ct=40). We blind-tested more samples and obtained very similar results with clear discrimination among different genotypes (data not shown). Results obtained from using molecular beacons were in complete agreement with results generated by conventional sequencing methods.
Stratagenes custom-designed molecular beacons are ideal for single nucleotide polymorphism genotyping. SNP genotyping requires only prior knowledge of a single nucleotide change within a target sequence and the design of a corresponding molecular beacon. With these requirements met, the subsequent molecular beacon procedure takes a quick 2 hours and is easily carried out in a 96-well format for large-scale screening. Additionally, using different molecular beacons labeled with different fluorophores to target different SNPs should make it possible to detect multiple polymorphisms5 simultaneously .
Real-time monitoring of polymerase chain reaction: Molecular beacons were synthesized by Stratagene. A detailed protocol for molecular beacon design is available on Stratagenes web site at http://www.stratagene.com/index.asp?catID=17. The nucleotide sequence of the molecular beacons was as follows: SNP11A: 5-fam-gcgcagagacggAacttgctctgaaactgcgc-DABCYL-3; SNP11G: 5-fam-gcgcagagacggGacttgctctgaaactgcgc-DABCYL-3. Underlined regions identify the probe sequences, and an A or G capital letter in bold indicate the A/G polymorphism. Primers FP 5-aacttcacctccccacatca-3 and RP 5-atctggggcacagaagcatt-3 (Research Genetics) were selected from the region flanking the SNP11 in the BAC and generated a 144-bp-long amplicon. Human genomic DNAs were isolated from peripheral white blood cells, and extractions were carried out with the Super QUIK-GENE DNA isolation kit (Analytical Genetic Testing Center). Each 50-ml PCR reaction contained 100 ng of human genomic DNA, 500 nM molecular beacon A or G, 400 nM of each primer, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.4 mM dUTP, 3.5 mM MgCl2, 2.5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems), and 1 U of uracil-N-glycosylase (UNG). Reactions were performed in triplicate in PE Applied Biosystems GeneAmp(r) 5700 Sequence Detection System. The PCR conditions were as follows: Cycling was preceded by 2 minutes at 50C; 10 minutes at 95C, followed by 40 cycles of 15 seconds; and 1 minute at 60C. In addition, a dissociation curve was generated after the last cycle of the PCR reaction. The thermal protocol for dissociation is defined as a hold at 95C for 15 seconds, a hold at 60C for 20 seconds, and a slow ramp (20 minutes) f rom 60C to 95C.
DNA sequencing: We amplified fragments with PCR primers flanking the 300- to 600-bp BAC genomic sequence of interest in four family members, ran the PCR products in agarose gel electrophoresis, and purified the PCR fragments (QIAGEN Inc). Sequencing was performed with fluorescent dideoxy dye-primer on an ABI Prism 377 sequencer, and all results were visually inspected.