A rapid, microplate SNP genotype assay for the leptinob allele.

A nonsense mutation in the mouse leptin gene causes genetic obesity. As a result of extensive research in the field of obesity, the use of leptinob mice is widespread. This mutation renders mice sterile, creating the need to breed heterozygous mice. For this reason, leptinob genotyping is necessary. To date, gel-based assays have been used for genotyping. Using the Invader Plus assay for single nucleotide polymorphism (SNP) detection, we have developed a gel-free microplate SNP assay for genotyping leptinwt and leptinob alleles.

Leptin is a 167 amino acid peptide hormone produced primarily in adipose tissue. It binds to a cell surface receptor, where it elicits a variety of physiological responses. It is a potent suppressor of appetite when it signals in the hypothalamus.
Leptin was discovered by positional cloning of a locus underlying the morbid obesity of the obese mutant mouse (1), a spontaneously occurring mutant that was discovered at the Jackson Laboratory in 1950 (2). This mouse has a nonsense mutation in the leptin gene, leading to a null allele. The obese mouse is widely used in studies of obesity and diabetes. In fact, it is the most widely used animal model of obesity in the entire Jackson Laboratory collection.
The pioneering studies of Coleman and others (3)(4)(5) showed that certain mouse strains become diabetic when made obese through the introgression of the leptin ob allele. These studies have inspired numerous investigators to combine the leptin ob allele with other mutations to gain new insights into the molecular, genetic, and physiological processes underlying resistance and susceptibility to obesity-induced type 2 diabetes (6,7). This strain is also used extensively in nutritional and pharmacological studies aimed at the treatment of obesity and diabetes.
The leptin ob allele has a CYT mutation that changes an arginine codon to a stop codon at position 105 (1). At present, the wild-type and mutant alleles are most commonly genotyped by restriction endonuclease digestion of a PCR-amplified sample of genomic DNA, followed by agarose gel electrophoresis (8,9). This procedure takes ?8 h to perform, of which ?1 h involves hands-on time.
Because of the need for gel electrophoresis, the length of this procedure increases considerably with the number of samples. An alternative method, single-stranded conformational polymorphism analysis, also involves gel electrophoresis and is quite labor-intensive (10).
We have developed a genotyping assay for leptin wt and leptin ob alleles that eliminates the need for gel electrophoresis, using the Invader Plus: assay for single nucleotide polymorphism (SNP) detection (11). The presence of the targeted SNP yields a fluorescent signal that can be quantitated in a standard plate reader. The entire process takes 0.5 h hands-on and a total of 2.5 h and can be scaled to hundreds of samples with minimal additional time.

Genomic DNA extraction
One centimeter tail clips from mice were digested overnight at 55jC in 300 ml of tail buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 25 mM EDTA, 0.5% SDS, and 0.1 mg/ml proteinase K). The digests were brought to room temperature, and 100 ml of Gentra Puregene Protein Precipitation Solution (Qiagen) was added. Samples were vortexed for 5 s and incubated on ice for 20 min. Samples were spun in a microcentrifuge at 4jC for 5 min at maximum speed. Supernatants were poured into a new tube containing 300 ml of isopropanol. Samples were inverted 20 times, or until a string of precipitated DNA was visible. Samples were spun again at room temperature for 2 min. Supernatants were poured off, and 300 ml of 70% ethanol was added. Tubes were gently inverted once and spun again at room tem-perature for 2 min at maximum speed. Again, the supernatants were poured off, with care to not lose the DNA pellets. Samples were spun at room temperature for 2 min at maximum speed. The supernatants were removed with a pipette tip, and the samples were air-dried for 30 min at room temperature. After 30 min, 200 ml of 13 Tris-EDTA was added to each pellet, and samples were stored overnight at 4jC before use. In preparation for the Invader Plus: assay, genomic DNA samples were first diluted on a 96-well plate: 4 ml (?200 ng to 1 mg of DNA) of genomic DNA into 120 ml of water (final volume, 303). Samples were carefully mixed by pipetting to avoiding cross-contamination of wells.

DNA sequences
All sequences are shown 5 ¶ to 3 ¶. The target DNA sequences for leptin (NM_008493) surrounding the SNP are GATGGA-GGAGGTCTCgGAGATTCTCCAGGTCATTGGT for leptin wt and GATGGAGGAGGTCTCaGAGATTCTCCAGGTCATTGGT for leptin ob , with the SNP nucleotide in lowercase. Primer sequences used to amplify this target region are Forward (CAGGTCCTC-ACCAGCCTGCCTTCC) and Reverse (CAGGGAGCAGCTCT-TGGAGAAGGCC). The Invader: oligonucleotide sequence is AATGACCTGGAGAATCTCA. The Invader: probe sequences are as follows: for leptin wt , gacgcggagCGAGACCTCCT; for leptin ob , cgccgaggTGAGACCTCCT, with the 5 ¶ flap region in lowercase.  leptin ob/ob , and leptin ob/1 known genomic DNA and three no-target controls were added. The plate was run in a thermal cycler with the following protocol: 2 cycles of 95jC for 2 min, 65jC for 1 min, and 72jC for 1 min; 33 cycles of 95jC for 15 s, 65jC for 1 min, and 72jC for 30 s; and one cycle of 99jC for 10 min, 50jC for 15 min, and 10jC hold.

Invader Plus: Assay
The plate was read on a Tecan Ultra 384 fluorescent plate reader with multilabeling capability. To detect the leptin ob allele, a fluorescein (FAM) excitation/emission wavelength of 485/ 535 nm was used, and for the leptin wt allele, a Red excitation/ emission wavelength of 570/620 nm was used. Data are represented as relative fluorescence units (RFU). A z optimization was initially done to determine the best z position and mirror setup for each excitation/emission wavelength.

Calculations
Data were analyzed according to the following calculation:

RFU Redsample RFU Redavergenotargetcontrol 21
If the FAM/Red ratio is ,0.5, the genotype is leptin 1/1 . If the ratio is .0.5 but ,15, the genotype is leptin ob/1 . If the ratio is .15, the genotype is leptin ob/ob .

RESULTS
We developed two genotyping assays, for the leptin wt and the leptin ob alleles. Two primers were designed to amplify the target region containing the SNP. One Invader: oligonucleotide was made that is complementary to the target region 5 ¶ of the SNP while containing a mismatch at the terminal 3 ¶ end. Two Invader: probe oligonucleotides were made, one containing the leptin wt nucleotide and one containing the leptin ob nucleotide. These probe oligonucleotides are complementary to the target region at the SNP nucleotide and 3 ¶ of the SNP, but they are not complementary 5 ¶ of the SNP. Instead, this 5 ¶ flap is complementary to a specific FRET cassette. During the 50jC incubation at the end of thermal cycling, the Invader: oligonucleotide and the probe oligonucleotide anneal to their complementary target DNA, creating a mismatch triplex of DNA at the SNP nucleotide (Fig. 1). This DNA triplex is recognized and cleaved by Cleavase: 3 ¶ of the SNP nucleotide, releasing the 5 ¶ flap of the probe oligonucleotide containing the SNP nucleotide at the terminal 3 ¶ end. Allele-specific 5 ¶ flaps will be released for each leptin wt or leptin ob allele. These 5 ¶ flaps will anneal to their specific FRET cassettes, creating another mismatch triplex of DNA, creating a substrate for Cleavase:. Cleavage releases the quencher from the fluorophore. Applying thermal cycling, the reaction and signal are amplified. We used a FRET cassette with Red fluorophore for the C nucleotide (leptin wt allele; Fig. 1A) and a FRET cassette with FAM fluorophore for the T nucleotide (leptin ob allele; Fig. 1B). The assay measures all three possible genotypes: Red signal only (leptin 1/1 ), FAM signal only (leptin ob/ob ), and both Red and FAM signals (leptin ob/1 ).
We obtained leptin 1/1 , leptin ob/ob , and leptin ob/1 mice (n 5 7 in each group) from our breeding colony. They were genotyped using single-stranded conformation polymorphism analysis. The samples were then subjected to the Invader Plus: assay, and the fluorescence was detected on a plate reader (Fig. 2). The FAM RFU corresponding to the leptin ob allele are shown in Fig. 2A. The Red RFU corresponding to the leptin wt allele are shown in Fig. 2B. The FAM/Red ratios corresponding to the genotype are shown in Fig. 2C.The mean FAM/Red ratios for the leptin 1/1 , leptin ob/1 , and leptin ob/ob mice were 0.07 6 0.02, 1.4 6 0.2, and 41 6 10 (P , 0.0001 by one-way ANOVA). Ratio thresholds were determined empirically by evaluat- ing the lowest ratio for leptin ob/ob and the lowest ratio for leptin ob/1 (Fig. 2C). There were no ambiguous results within the samples tested. These ratio thresholds can change if there is a differential decrease in fluorescence signal strength. This can occur with time and light exposure. An Excel template was used for ease in the analysis of genotypes. This template and a detailed protocol are available in the supplementary material.

DISCUSSION
We have developed and validated a gel-free, microplate fluorescence-based genotyping assay for the wild-type and obese alleles of leptin. This method is extremely reliable, is able to identify heterozygotes unambiguously, and is quite rapid. The method is amenable to automation and can easily be scaled to perform large numbers of assays in parallel. When this method was used to test our unknown samples, the failure rate was ,1%. A failure will not result in a genotyping error because it results in the absence of both the FAM and Red signals. A single plate can handle 90 samples plus controls. Because of the extensive use of leptin ob mice, this method is an invaluable tool.