Methods

GTCA Testing Methods

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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.

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.
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.

Hydrolysis Probes

For Quantitative PCR, we utilize a multiplexed reaction containing the primers and probes designed for a specific region of the target and the primers and probes for a housekeeping gene which is present in the wild-type genome. The probe is designed to sit down on the DNA in the region between the specific primers. As the polymerase copies the DNA in the PCR reaction the 5′-3′ exonuclease activity of Taq Polymerase cleaves the probe in a hydrolysis reaction releasing the reporter fluorophore and the quencher. They allow the fluorophore to give off light at a wavelength specific to that fluorophore. We utilize probes that are labeled with FAM for the internal control amplicon and labeled with HEX for the mutant product. These are detected at different wavelengths in the reaction. By collecting data as the samples amplify, we can generate amplification curves as shown below for the FAM probe in this transgenic assay.

In the graph below, the red horizontal line is the “Noise band.” We use the point where the amplification crosses this red line as the CP (crossing point) which is also called the CT point in other manufacturers software. We then compare the CP of the sample for the internal control and the CP of the transgene target to determine zygosity. In the graph of this differential CP, we see samples that are hemizygous for the transgene as about 1 CP above that of samples that are homozygous. Utilizing the internal control compensates for differential concentrations of the starting samples.

Endpoint Analysis

Hydrolysis probes for the detection of SNPs and Indels

The mechanics of using hydrolysis probes for the detection of SNP’s and Indels is identical to that used to detect transgene zygosity with a few exceptions. For the most part, we are looking for a small genetic change in the mouse genetic code. We place primers on either side of the change and amplify a small amplicon that includes the mutation. Included in the reaction is a FAM labeled probe and a HEX labelled probe. One is an exact match for the wild-type sequence at the site where the mutation occurs. The other is an exact match for the mutant sequence at the site of the mutation.

Post-amplification the relative amount of destroyed FAM and HEX probe is graphed. In most cases, the allele along the X axis is the wild-type allele (blue=Wild), the allele along the Y axis is the mutant allele (green=Hom) and the allele along a 45-degree angle is a 50/50 mixture of wild-type and mutant allele (red=Het).

HRM and Gel Electrophoresis

High resolution melting (HRM) is a technique used to differentiate two products that cannot easily be separated using normal melt curve techniques. The technique was originally developed to identify unknown SNPs in a sequence of interest. GTCA primarily uses this technique to detect floxed alleles where we do not have enough information to create allele specific assays. The sequence of a loxP site and any attending additional sequence that is inserted with the loxP, typically about 100 bp, is usually of very low GC content. This makes the difference hard to see using melt curve analysis. Below is an example of an assay that we perform HRM analysis on normally.

The first image is of the samples using melt curve analysis. We have found that using HRM analysis for this type of product is far more reliable.

The second image is our HRM analysis of the same samples. The HRM analysis allows for clear and unambiguous calls of the genotypes. In this example the blue lines are wild-type, red are heterozygous and green are homozygous.

The third image is the difference plot of the HRM analysis. Again, blue are wild-type, red are heterozygous and green are homozygous. The use of known controls is vital to this technique.

Some floxed alleles are not amenable to HRM analysis. In these rarer cases, we must perform gel electrophoresis of the samples to determine the size variation in the products. All products are checked on melt curve to determine that the samples and controls have amplified prior to gelling. A lack of amplification in the NTC is also confirmed. We only gel one replicate of the samples and the need to gel the samples post amplification can increase the turnaround time by 1 day.

Melt Curve Analysis

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.
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.

Hydrolysis Probes

KASP

KASP assays are designed by LGC Genomics

The Kompetitive Allele Specific PCR genotyping system (KASP™) is a homogeneous, fluorescent, endpoint genotyping technology offered by LGC Genomics. KASP uses three components: test DNA with the SNP of interest; KASP Assay mix containing two different, allele-specific, competing forward primers with unique tail sequences and one reverse primer; the KASP Master mix containing FRET cassette plus Taq polymerase in an optimised buffer solution. KASP assays are designed by LGC Genomics. Primer sequences are not provided for any KASP assay.

HRM and Gel Electrophoresis

The first image is of the samples using melt curve analysis. We have found that using HRM analysis for this type of product is far more reliable.

The third image is the difference plot of the HRM analysis. Again, blue are wild-type, red are heterozygous and green are homozygous. The use of known controls is vital to this technique.