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About SNP Genotyping

The Molecular Genomics Core (MGC) offers Taqman-bound allele discrimination analysis as a service.

Samples should be submitted in the form of genomic DNA either frozen or as an ethanol precipitate.  Once received, sample quantity will be determined via the NanoDrop which will be used to dilute the sample to the appropriate concentration for processing. 

Genotyping Services

  • Taqman-Based Allelic Discrimination

Service Features & Options

  • Experimental Design
  • Assay design & optimization (TaqMan/DNA Seq)
  • Sample Quantitation via NanoDrop® ND-1000
  • Data Analysis

Useful Links
SPLAT User’s Manual (PDF)
ABI’s TaqMan SNP Overview (PDF)
TaqMan SNP Genotyping Assay (PDF)

To schedule an appointment or to discuss experimental design, send an email to Debbie Prusak or Dr. Tom Wood.

SNP Genotype Service Information

About TaqMan-based SNP Genotyping

Taq DNA polymerase’s 5’-nuclease activity is used in the Taqman assay for SNP genotyping. The Taqman assay is performed concurrently with a PCR reaction and the results can be read in real-time as the PCR reaction proceeds. The assay requires forward and reverse PCR primers that will amplify a region that includes the SNP polymorphic site. Allele discrimination is achieved using FRET combined with one or two allele-specific probes that hybridize to the SNP polymorphic site. The probes will have a fluorophore linked to their 5’ end and a quencher molecule linked to their 3’ end. While the probe is intact, the quencher will remain in close proximity to the fluorophore, eliminating the fluorophore’s signal. During the PCR amplification step, if the allele-specific probe is perfectly complementary to the SNP allele, it will anneal to the target DNA strand and then get degraded by the 5’-nuclease activity of the Taq polymerase as it extends the DNA from the PCR primers. The degradation of the probe results in the separation of the fluorophore from the quencher molecule, generating a detectable fluorescent signal. If the allele-specific probe is not perfectly complementary, it will have a lower melting temperature and not anneal as efficiently. This prevents the nuclease from acting on the probe.

TaqMan-based genotyping is most appropriate for projects of medium size (100-1000 samples) being genotyped for a small number of SNPs (1-50). It is possible to use it with smaller size projects although DNA sequencing might be more economical in this case.  The TaqMan service begins with a review of the list of SNPs to be genotyped as not all SNPs can be typed using the TaqMan technology. Once your list of SNPs is finalized we will custom design or if available order an Assays-On-Demand (AOD) SNP assay from Applied Biosystems. While awaiting receipt of your assays, we will assess the quantity of your DNA using either a NanoDrop or PicoGreen assay. We require 30ng of genomic DNA per SNP assay and DNA must be supplied at 30ng/ul in 96 well plates.  All samples must be at the same concentration and purity for the procedure to give best results. Purity should be assessed spectrometrically and a ratio of OD260/OD280 equal to 1.8.  Our facility runs the standard TaqMan protocol as recommended by the manufacturer, which is successful for the majority of the SNP assays. However, you may see a decrease in the call rate for a small number of "difficult" assays.

To schedule an appointment or to discuss experimental design, send an email.

Q: How much total DNA do I need to supply?
A: We recommend submitting 1ug or more of gDNA.

Q: How do you isolate genomic DNA and from what sources?
A: Routinely, we isolate genomic DNA from blood, tissue (plant/animal/microbial) and cell culture using SDS and proteinase K treatment and phenol/chloroform extraction. This procedure yields the high quality DNA that is ideal for labeling, restriction digests and DNA sequencing, and genomic libraries, and other molecular biology steps.

Q: How long will it take to get my results back?
A: Samples are handled on a first come first serve basis. It is recommended that you contact the MGC in the early stages of planning an experiment so that we can anticipate the samples arriving to us for processing.

Q: Are you able to assist with data analysis?
A: Yes, please contact us for more information at genomics.core@utmb.edu

 

Q: Are you able to assist with the Experimental design of My project?
A: All investigators who wish to pursue a project through the core facility are strongly encouraged to meet with the Genomics Core Staff to ensure proper experimental design and a clear understanding of sample requirements for processing through the facility.

To schedule an appointment or to discuss experimental design, send an email.

Allele  One of a number of different forms of a gene. Each person inherits two alleles for each gene, one allele from each parent. These alleles may be the same or may be different from one another.

Recessive  A gene that is phenotypically manifest in the homozygous state but is masked in the heterozygote by the presence of a dominant allele.

Dominant  Refers to a characteristic that is apparent even when the relevant gene is present in only one copy. A dominant disorder can be inherited from only one parent.

Penetrance  The probability of expressing a phenotype given a genotype. Penetrance is described as either"complete"or"incomplete."For example, individuals who carry the gene for tuberous sclerosis have an 80% chance of expressing the disorder. Penetrance may also be dependent on a susceptible individual's current age. For example, 20% of all gene carriers for myotonic dystrophy express the gene to some degree by age 15 while 80% of all gene carriers express it by age 60. ...

Epigenetic  A factor that changes the phenotype without changing the genotype.

SNP  Single necleotide polymorphism, or a single nucleotide position in the genomic sequence for which two or more alternative alleles are present at appreciable frequency (traditionally, at least 1%) in the human population.

Copy #  The average number of molecules of a plasmid or gene per genome contained in a cell.  (Loss or Gain)

Rearrangement  A change in the usual order and arrangement of genetic material either within the chromosome complement or within a gene locus.

Translocation  A rearrangement occurring when a piece of one chromosome is broken off and joined to another chromosome. An individual with a balanced translocation has the normal amount of chromosomal material. A person with an unbalanced translocation will have a loss or gain of chromosomal material.

Loss of Heterozygosity (LOH)  A situation where one chromosome has a normal allele of a gene and one chromosome has a mutant or deleted allele.

Ploidy  The number of (complete) sets of chromosomes in a cell or an organism. For example, haploid means one set and diploid means two sets.

Uniparental disomy  When both members of a chromosome pair are contributed by one parent rather than one from each parent. Uniparental disomy may be maternal or paternal.

Tumor suppressor genes  The role of tumor suppressor genes is to maintain the integrity of the DNA in cells. The activation of a tumor suppressor gene at certain "check points" puts on the "brakes" and allows the cell to check for any damage in its DNA. Usually repairs are made before the cell is allowed to go on and divide. Without these brakes, cells with damaged DNA would copy the damage and pass damage on to daughter cells. The damage becomes a permanent mutation in future generations of new cells.  Tumor suppressor genes are recessive.

To schedule an appointment or to discuss experimental design, send an email, or call (409) 772-6349.

  Shipping Address

  Molecular Genetics Facility
  6.158 Medical Research Building
  11th & Mechanic
  Galveston, TX 77555-1079

Sample Drop-Off

  M-F, 8am - 5pm
  6.160 Medical Research Bldg

Contact

  (409) 747-0387
  Genomics.core@utmb.edu