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DNA Array Core Facility

Figure 1: Schematic representation of mutation detection using melting curve analysisRoche LightTyper

Genotyping services provided by the DNA Array Core Facility utilizes melting curve analysis to determine wild-type sequence, heterozygotes, and homozygous single nucleotide polymorphisms (SNPs). Single base mismatches between a labeled probe and the amplicon containing the target polymorphism exhibit reduced melting temperatures detected by the Roche Applied Sciences LightTyper instrument.

  

Overall Scheme:

  • Fluorescently labeled sequence-specific oligonucleotide probe is added to the PCR reaction
  • Amplification of target polymorphism by PCR
  • Generation of a melting curve by slowly heating the amplicon/probe heteroduplex and measuring the changes in fluorescence as the probe denatures from the amplicon
  • Genotype calls made based on melting curves generated (Figure 1)

Procedure:

  • PCR primers and probes are designed using the LightTyper Primer and Probe Design Software 1.2.0. This software designs primer/probe sets that are optimized to maximize the difference in melting temperature between the perfectly matched and mismatched probes
  • MgCl concentrations and annealing temperature conditions are optimized for the PCR reaction
  • Fluorescein labeled probes are added to the PCR reactions set-up in 96 well plates
  • After PCR the plate is loaded onto the LightTyper instrument's thermal block. The plate is slowly heated while fluorescence is constantly measured by a high-resolution CCD camera
  • Using the LightTyper Analysis Software genotype calls can be made based on the melting peaks generated. The software is also capable of automatically calling genotypes based on supplied standards and thresholds. Post-PCR to genotyping results can take as little as 10 minutes. (Figure 2)

Figure 1: Schematic representation of mutation detection using melting curve analysis

Figure 1: Schematic representation of mutation detection using melting curve analysis

Figure 2: Screen capture from LightTyper Analysis Software

Figure 2: Screen capture from LightTyper Analysis Software displaying melt curves for representative genotype groups

Sample Requirements:

Minimum 1 ug of DNA at a concentration of 10 ng/ul in T.E. (10mM Tris pH 7.5-8.3, 1mM EDTA). Preferably DNA samples should be provided in 96 well plates but DNA in tubes will also be accepted.

Service Charges:

Call the DNA Array Core Facility at (858) 784-2263 for detailed information regarding charges for genotyping and other related costs.