SNP genotyping was performed by using high-throughput MALDI-TOF mass spectrometry. Primers and probes were designed by using the SpectroDESIGNER software (Sequenom, San Diego). Multiplex PCRs were performed, and unincorporated dNTPs were dephosphorylated by using shrimp alkaline phosphatase (Hoffman-LaRoche, Basel) followed by primer extension. The purified primer extension reaction was spotted onto a 384-element silicon chip (SpectroCHIP, Sequenom) and analyzed in the BrukerBiflex III MALDI-TOF SpectroREADER mass spectrometer (Sequenom), and the resulting spectra processed with SpectroTYPER (Sequenom). Sequence characterization of these genes revealed 38 SNPs across the targeted regions and was genotyped through sequenome in the given sample of goats. The gene and genotypic frequencies of SNPs in studied genes have been presented in Table 1 .
Spectrotyper
Manufactured by Labcorp
The SpectroTYPER is a laboratory instrument used for spectral analysis. It is designed to accurately measure and identify the spectral characteristics of various samples.
Automatically generated - may contain errors
Lab products found in correlation
Shrimp alkaline phosphatase, by Roche (2 mentions)
Spectrochip, by Labcorp (2 mentions)
Spectrodesigner software, by Labcorp (2 mentions)
Iplex enzyme, by Labcorp (1 mentions)
Massarray platform, by Labcorp (1 mentions)
Ezna blood dna midi kit, by Omega Bio-Tek (1 mentions)
Hotstartaq dna polymerase, by Qiagen (1 mentions)
3 protocols using spectrotyper
1
SNP Genotyping in Goat Samples
2
SNP Genotyping Using MALDI-TOF Mass Spectrometry
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A total of 20 cc of blood was collected from each participant. Genomic DNA was isolated from peripheral lymphocytes using the phenol/chloroform extraction method. SNP genotyping was performed using high-throughput matrix-assisted laser desorption and ionization–time of flight (MALDI-TOF) mass spectrometry. Briefly, primers and probes were designed using SpectroDESIGNER software (Sequenom, San Diego, California, USA). Multiplex PCRs were performed, and unincorporated ddNTPs were dephosphorylated using shrimp alkaline phosphatase (Hoffman-LaRoche, Basel, Switzerland), followed by primer extension. The purified primer extension reaction was spotted onto a 384-element silicon chip (Spectro-CHIP, Sequenom) and analyzed using an autoflex MALDI-TOF SpectroREADER mass spectrometer (Sequenom); the resulting spectra were processed with SpectroTYPER (Sequenom). The people who performed the genetic study were blinded to the clinical data of the study subjects.
3
Genotyping of Blood Leukocyte SNPs
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Genomic DNA was extracted from blood leukocytes using an EZNA TM Blood DNA Midi Kit (Omega Bio-Tek, Norcross, GA, USA), according to the manufacturer instructions. Genotyping for the eight SNPs was performed using the MassARRAY platform (Sequenom, San Diego, CA, USA) by means of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, according to the manufacturer instructions. Primers were designed using the Sequenom software (Table 1), and the extension reaction produced allele-specific products with masses differing by 30 Da, or approximately one single nucleotide. Primer extension and polymerase chain reaction were performed according to the manufacturer instructions using an iPLEX enzyme (Sequenom) and HotStarTaq DNA polymerase (Qiagen, Hilden, Germany). Genotypes were automatically identified by the SpectroTYPER software (Sequenom), and only conservative and moderate calls, as defined by the software, were accepted for this study.
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