> Services
Genotyping services provided by the Hartwell Center to St. Jude investigators and their staff currently include the analysis of Minisatellites and Microsatellites, screening for genomic insertions and deletions, and multiplexed Single Nucleotide Polymorphism (SNP) screening, copy number detection and methylation quantification, DNase Footprinting, Surveyor low-frequency mutation detection, Restriction Fragment Length Polymorphism and other commercialized assays.
The technique used for the genotyping of Minisatellites and Microsatellites, the screening of insertions and deletions, and SNP screening utilizes the fluorescence detection capabilities of our Applied Biosystems 3730xl Genetic Analyzer. PCR amplification primers are constructed by our DNA synthesis facility with a fluorescent tag on the 5' end of the forward primer. PCR amplification of target DNA with these primers creates amplicons that contain the specified fluor. Aliquots of these PCR products are then separated by capillary electrophoresis on the 3730xl Genetic Analyzer and their sizes calculated by comparison to a concurrently run size standard.
The resulting run files created during the capillary electrophoresis separation are loaded into our analysis software, GeneMapper 4.0 from Applied Biosystems, for automated allele calling and genotype quality scoring. Any allele calls that do not meet defined analysis parameters are flagged and reviewed by Hartwell Center staff to insure the highest quality data for our users. When the data analysis is completed a text file containing the resulting genotypes will be electronically sent to the specified user. All data is placed in our archive for secure long term storage and can be retrieved by request.
Additional information is available for each of the current genotyping services. Please contact Dr. Jiang Li (Jiang.li@stjude.org or X4842) for assay design.
- Minisatellite
and Microsatellite Analysis
Assays are currently available for a number of Minisatellites and Microsatellites located throughout the genome. Special consideration has been given to those markers associated with the Minisatellites and Microsatellite Instability phenotype in various diseases. Additional assays will be added at the request of St. Jude Investigators. - Insertion/Deletion
Screening
Fragment Size Analysis is currently being used to screen for specific insertions and deletions of interest to St. Jude investigators. This technique is extremely accurate and can be used to screen a variety of these types of genomic events. Assays for specific polymorphism's are designed and validated at the request of St. Jude investigators. - Mouse
Genotyping
Many of the various transgenic constructs utilized by St. Jude investigators and their staff are readily amenable to automated screening by our genotyping facility. Each construct requires us to validate a detection assay that will then be made available to the investigator indefinitely. - SNP Screening
SNP Multiplexing assays including ABI SNaPshot and ABI SNPlex are currently being used in screening SNPs. ABI SNaPshot Multiplex System allows multiplexed single base extension of up to 10 SNPs and generates genetic variation data in a few simple steps. It uses inexpensive, unlabeled primers interrogate multiple SNPs regardless of chromosome position and enables separation of SNP loci down to one base-pair. ABI SNPlex Technology is based on Oligonucleotide Ligation Assay (OLA) and hybridization of amplified products to ZipChutes (universal reporter probes). ZipChutes are eluted then loaded onto the 3730XL DNA Analyzer. ZipChutes are tagged with mobility modifiers which allow separation along a microcapillary in a dense multiplex. Currently SNPlex allows multiplexed OLA of up to 48 SNPs. Different issues can influence platform selection and assay design, such as the number of SNP and the composition of the surrounding genomic region. In silicon-based assay design and ABI SNPBrowser are currently being used to help investigators for SNP assay design. Specific requests from investigators to design an assay for SNPs which are important to their research are always giving priority. -
Multiplex Ligation-dependent Probe Amplification (MLPA)
MLPA is a method to evaluate the copy number of up to 45 nucleic acid sequences in one single reaction. The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences as well. MLPA can be applied on SNP genotyping, copy number detection, methylation quantification as well as mRNA profiling. Amplification products are separated by capillary electrophoresis. MLPA is considered as a customized SNPlex assay with a high cost-efficiency. We also provide a service of multiplexing assay design. - DNase Footprinting
DNase Footprint Assay is used to determine the binding site of a protein regulator to DNA which is typically in a promoter region. The protein of interest provides protection to specific nucleotides resulting in fewer DNA fragments in this area after the digestion step. The resulting fragment pattern can be analyzed with the capillary electrophoresis and compared to DNA sequencing reaction products in order to determine the exact sequence which interact with the protein. - PCR-Restriction Fragment Length Polymorphism (RFLP)
By designing primers that will introduce or destroy a restriction site for one of the alleles, the PCR products for SNP alleles can be distinguished by restriction fragment lengths. - Surveyor mutation assay
The goal is to screen multiple mutations with a low allele frequency. A new mismatch-specific plant DNA endonuclease is used to scan for known and unknown mutations and polymorphisms in heteroduplex DNA. The signal to noise ratio is generally high enough to detect mutations present as a low percentage of the total mutant and wild-type DNA. At present, 2.5% to 20% mutant DNA can be detected by capillary electrophoresis depending upon the particular DNA amplicon, its size, the number and types of mutation. - Promega Powerplex system
ATCC is developing a comprehensive database of DNA fingerprints of STR (short tandem repeat) loci for all of their human cell lines. The fingerprinting process used at ATCC involves simultaneously amplifying eight STR loci and the amelogenin gene in a multiplex PCR reaction (PowerPlex 1.2 system) which allows for discrimination of fewer than 1 in 108 individuals.