Methods

GTCA Testing Methods

Not all samples are amenable to genetic testing using the same methods.  To ensure you receive the most accurate results we employ a number of methods as appropriate for your samples.

Melt Curve Analysis

Assays are designed to amplify specific regions of the target sequence in the presence of SYBR green.  Post amplification, the amplicons are subjected to an increasing temperature while data is collected.  SYBR green intercalates with double stranded DNA and releases light at a wavelength detected by the instrument.  As the temperature increases the double stranded DNA will melt apart based primarily on two factors. 

  1. The length of the DNA amplicon. The longer the amplicon the more hydrogen bonds present and the greater the required energy to separate the strands.
  2. The G-C content. This is because G-C pairs share 3 hydrogen bonds, while A-T pairings only share 2 hydrogen bonds. So, the higher the G-C content the more energy, higher temperature, necessary to make the DNA single stranded.

When the DNA becomes single stranded, SYBR Green no longer intercalates with the DNA and the wavelength of light released shifts out of the range of the detector in the instrument.

To perform the testing, we amplify the DNA using primers which target specific regions in the DNA.  One target is a portion of the sequence in the transgene.  We also amplify a second target that is present in all mice.  This internal control is used to demonstrate that DNA was added to the reaction and that amplification could have occurred in the transgene was present.  The particular internal control is picked to not interfere with the detection of the transgene.  We also use this method to identify alleles in targeted mutations.  However, in targeted mutations the wild-type and mutant alleles are run in separate wells.

After the DNA is amplified, we bring all the samples to 70°C, then slowly raise the temperature.  When the temperature reaches a temperature at which the amplified DNA melts apart to become single stranded, the amount of fluorescence in the well decreases. As stated above, the temperature that the DNA melts apart is specific to the particular target that is amplified.  Below is a graph of a melt curve of a wild-type (red) and a transgene positive (green) sample. The software is then used to mathematically convert these melt curves to melting peaks as seen in the image to the right. Therefore, amplicons designed for this method can be differentiated based on the melt profiles of these different products.

Melt Curve Analysis

Assays are designed to amplify specific regions of the target sequence in the presence of SYBR green.  Post amplification, the amplicons are subjected to an increasing temperature while data is collected.  SYBR green intercalates with double stranded DNA and releases light at a wavelength detected by the instrument.  As the temperature increases the double stranded DNA will melt apart based primarily on two factors. 

  1. The length of the DNA amplicon. The longer the amplicon the more hydrogen bonds present and the greater the required energy to separate the strands.
  2. The G-C content. This is because G-C pairs share 3 hydrogen bonds, while A-T pairings only share 2 hydrogen bonds. So, the higher the G-C content the more energy, higher temperature, necessary to make the DNA single stranded.

When the DNA becomes single stranded, SYBR Green no longer intercalates with the DNA and the wavelength of light released shifts out of the range of the detector in the instrument.

To perform the testing, we amplify the DNA using primers which target specific regions in the DNA.  One target is a portion of the sequence in the transgene.  We also amplify a second target that is present in all mice.  This internal control is used to demonstrate that DNA was added to the reaction and that amplification could have occurred in the transgene was present.  The particular internal control is picked to not interfere with the detection of the transgene.  We also use this method to identify alleles in targeted mutations.  However, in targeted mutations the wild-type and mutant alleles are run in separate wells.

After the DNA is amplified, we bring all the samples to 70°C, then slowly raise the temperature.  When the temperature reaches a temperature at which the amplified DNA melts apart to become single stranded, the amount of fluorescence in the well decreases. As stated above, the temperature that the DNA melts apart is specific to the particular target that is amplified.  Below is a graph of a melt curve of a wild-type (red) and a transgene positive (green) sample. The software is then used to mathematically convert these melt curves to melting peaks as seen in the image to the right. Therefore, amplicons designed for this method can be differentiated based on the melt profiles of these different products.