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Fragment Analysis: General
In order to determine the reagents needed, it will be necessary to gather initial information such as the following:
- What is the minimum and maximum amplicon size range?
- Will there be one target per sample or multiple targets (singleplex versus multiplex on the capillary electrophoresis instrument)?
- Is there overlap among the size of amplicons?
This initial information will help identify the spectral calibration needed for the capillary electrophoresis instrument, the fluorescent dye used to label the primer, and the size standard. Additional information can be found in the “Experimental Design” section of the DNA Fragment Analysis by Capillary Electrophoresis Guide.
There are several fragment analysis applications that can be run on the CE instruments, such as:
microsatellite analysis, SNP genotyping, fingerprinting, and relative fluorescence quantitation. Please see the DNA Fragment Analysis by Capillary Electrophoresis Guide for more details about these applications.
The largest detectable size/resolution for fragment analysis is dependent on the capillary length, polymer and run module used on the instrument. We have run modules optimized to provide 120–1,200 bp sizes. Additional information regarding specific instruments and the resolution obtained with specific run modules can be found in Chapter 4 of the DNA Fragment Analysis by Capillary Electrophoresis Guide.
We provide POP™ Conformational Analysis Polymer (CAP) for non-denaturing polynucleotide fragment analysis applications on the Applied Biosystems™ 3130/3130xl Genetic Analyzers. However, we do not have a premade kit specifically for this application. To learn more about non-denaturing applications, please see here.
Beyond 1,200 bp, the peaks may not resolve well and sizing of these larger peaks may vary, as we do not have a size standard that goes above 1,200 bp. The capillary electrophoresis instrument run times and run voltage may need to be modified to collect fragments greater than 1,200 bp.
It is possible to use another company’s kit. However, the vendor should be contacted to determine if they have protocol(s) for their kit(s) on the specific capillary electrophoresis (CE) instrument (specifically, the model you intend to use), and if they have reagents to run a spectral calibration or matrix run on the CE instrument with compatibility to the dyes that are part of the kit(s) in question.
Primer Labeling and PCR Amplification
The fluorescent dyes are grouped into dye sets. The dye sets, corresponding matrix standards, and corresponding dyes are shown below:
C (310 only)
(ROX™, LIZ™, and TAMRA™ dyes are reserved for the size standard)
We recommend using only Applied Biosystems™ dyes, as we provide spectral calibration reagents that have been optimized for our dye sets. Non-Applied Biosystems™ dyes have variable emission spectra and also require a spectral calibration generated for the specific dyes in use to correct for the spectral overlap between the dyes. You are responsible for obtaining the appropriate spectral calibration reagents and for optimizing custom dye sets to ensure the dye labels do not affect PCR efficiency. The use of dyes outside of the recommended dye sets can result in pull-up and pull-down peaks, which may make allele calling challenging.
A dye set is a combination of dyes that have minimum spectral overlap, which will allow you to multiplex products of similar size using different dyes. Although the size ranges may overlap, the use of a different dye will allow the software to easily distinguish between these products.
Both DS-30 and DS-31 matrix standards contain 6-FAM™, NED™ and ROX™. The difference is the dye used in the green channel where DS-30 contains the HEX™ dye versus DS-31 which contains the VIC™ dye. The VIC™ dye emits a stronger signal and is more stable than the HEX™ dye and we suggest that you use the VIC™ dye for weak amplicons. Both DS-30 and DS-31 are contained in dye set D, as both dyes have excitation and emission wavelengths that are quite similar and, upon the initial release of DS-31 on the Applied Biosystems™ 310 Genetic Analyzer, it was not necessary to change the filter set to run DS-30 and DS-31.
We do not recommend that you switch dyes in the middle of a project. Switching dyes may result in a shift in the allele sizes as dyes differ in their mobility. If dyes are switched in the middle of a project, it will be necessary to identify the size shift for all the markers/targets in the amplicon product range. The size shift will be consistent throughout the project and it is only necessary to make the adjustment once.
It is possible to amplify multiple targets within the same PCR reaction. However, there are limitations such as primer-oligomer formation, loss of specificity, and decreased yield of specific products which will require a significant amount of optimization to overcome. We recommend that you perform the PCR reactions in singleplex and then pool the PCR products prior to loading on the capillary electrophoresis instrument.
The number of PCR products that can be pooled is determined by the size range of the PCR products and whether 2, 3 or 4 dyes in the same dye set are used for labeling the primers. PCR product sizes may overlap, but it is necessary to use different fluorescent dyes so they can be distinguished from each other.
The quantity of PCR product is determined empirically. Typically 0.5 µL of PCR product is used. However, the PCR product may be from a dilution of 1:2, 1:5, 1:10 or greater. The PCR product may require additional dilution to prevent overloading of the capillaries and saturation of signal on the CCD camera. Please refer to Chapter 4 of the DNA Fragment Analysis by Capillary Electrophoresis Guide for additional recommendations on sample loading concentration and optimizing signal intensity.
The use of labeled primers does not typically require the need for further optimization of the PCR reaction. Some optimization may be needed regarding dilution of the PCR product on the capillary electrophoresis instrument to prevent overloading of the capillaries and saturation of signal on the CCD camera. Please refer to Chapter 4 of the DNA Fragment Analysis by Capillary Electrophoresis Guide for additional recommendation on sample loading concentration and optimizing signal intensity.
For long-term storage (greater than a week), storage at-20 degrees C is recommended. However, for short-term storage (less than a week), 4 degrees C storage can be used
Stutter peaks are artifacts of the PCR reaction and occur most commonly with mono-, di-, tri-, and tetranucleotide repeats. They may be caused by polymerase slippage during elongation. Stutter peaks appear as multiple lower peaks that precede the true allele peak. These stutter peaks differ in size from the true allele peak by multiples of the length of the repeat unit.
Non-templated A addition is an artifact of the polymerase. It occurs about 50% of the time, and for fragment analysis applications, will result in 2 peaks, one base pair apart for one allele. This may result in genotyping errors and require manual editing of the allele calls. It can be prevented by using a high-fidelity, proofreading polymerase. There are also options to promote plus A addition, one commonly used option being use of additional sequence, commonly referred to as a tail, on the unlabeled primer.
Primers can be reconstituted in sterile 1X TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0), sterile 1X low TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0), or molecular-grade, nuclease-free water. For more information on how to dilute your custom primers, refer to the following document.
A stock concentration can be between 10 µM and 100 µM. A working concentration can then be made depending on the concentration needed in the PCR reaction.
The oligos are good for one year from the date of receipt when stored at -20 degrees C.
Fluorescently-labeled oligos should be protected from light to prevent photobleaching. They should be wrapped in aluminum foil or kept in a cardboard box in a -20 degrees C freezer.
To minimize freeze/thaw cycles, we recommend you aliquot the stock dilution into small “working” volumes, according to the number of reactions, and store at -20 degrees C. Prior to use, the solution should be lightly vortexed and spun down using a bench-top centrifuge.
The tail option is used to promote 3´ A nucleotide addition to the (forward) labeled strand. DNA polymerases catalyze the addition of a single nucleotide (usually an adenosine) to the 3´ ends of the two strands of a double-stranded DNA fragment. This non-template complementary addition results in a denatured PCR product that is one nucleotide longer than the target sequence. Because 3´ A nucleotide addition rarely goes to completion without a long extension step at the end of thermal cycling (i.e., only a fraction of the fragments receive the extra nucleotide), single-base ladders often form and the resulting allele calls can be inconsistent, incorrect, or missing entirely, forcing you to inspect all allele calls and to correct erroneous calls manually.
For the majority of fragment analysis applications, desalted primers are fine. For SNaPshot™ primers, HPLC-purification will be necessary.
The choice of size standard is determined by the minimum and maximum range of the amplicons. Select a size standard with at least two sizing fragments smaller and larger than your unknown sample fragments, and with a dye that is compatible with the dyes used for labeling the primers in the dye set calibrated on the instrument. Please see the table below:
|GeneScan™ LIZ™ Dye Size Standard, 5-dye chemistry||GeneScan™ ROX™ Dye Size Standard, 4-dye chemistry|
Expected marker length in basepairs
Size standards stored at -20 degrees C can result in loss of the smaller size standard peaks, lower signal, or no signal. The size standard should be run with HiDi™-Formamide only to determine the impact to the size standard peak intensity.
The size standard expiration date can be found either on the original box/pouch or the Certificate of Analysis (COA). To access the COA, click here and search with the Lot No. If there is no expiration date listed on the box or COA, the warranty date is 1 year from date of receipt.
The choice of size standard is determined by the minimum and maximum range of the amplicons. Select a size standard with at least two fragments smaller and larger than your unknown sample fragments, and with a dye that is compatible with the dye set used for labeling the primers. For PCR products greater than 500 bp, either the GeneScan™ 600 LIZ™ Dye Size Standard or GeneScan™ 1200 LIZ™ Dye Size Standard may be used.
The GeneScan™ 500 LIZ™ Dye Size Standard and GeneScan™ 500 ROX™ Dye Size Standard differ in the fluorescent dyes. The GeneScan™ 500 LIZ™ Dye Size Standard is used with the 5-dye chemistry and the G5 dye set. The GeneScan™ 500 ROX™ Dye Size Standard is used with the 4-dye chemistry and the DS-30, DS-31 and DS-32 dye sets.
For fragments that are greater than 600 bp, the GeneScan™ 1200 LIZ™ Dye Size Standard is recommended. The GeneScan™ 1000 ROX™ Dye, or GeneScan™ 2500 ROX ™ Dye Size Standards are intended for non-denaturing applications only.
Switching size standards in the middle of a project may result in a shift in the allele sizes, as size standard dyes differ in their mobility. If the size standard change is done, it will be necessary to identify the size shift for all the markers/targets in the product. Switching from a LIZ™ dye to a ROX™ dye will also require running it under a new dye set. Make sure the dyes you are using are compatible with the dye set D and that you have a spectral for the new dye set.
If the targets of interest are greater than 600 bp, it is necessary to use the GeneScan™ 1200 LIZ™ run module. The GeneScan™ 1200 LIZ™ run module has reduced run voltage and increased run time to allow for collection of all of the GeneScan™ 1200 LIZ™ basepair fragments and also ensures the size standard peaks are linear in their migration. Not using the GeneScan™ 1200 LIZ™ run module may result in failed sizing analysis in GeneMapper™ software and loss of the larger peaks of the size standard.
Switching size standards in the middle of a project may result in a shift in the allele sizes, as size standard dyes differ in their mobility. If the size standard change is done, it will be necessary to identify the size shift for all the markers/targets in the product.
The SNaPshot™ Multiplex Kit uses the E5 dye set which contains the dR110, dR6G, dTAMRA™, and dROX™ dyes, and uses LIZ™ dye-labeled size standard.
Follow these recommendations for designing and evaluating primers:
- Primers included in a single reaction need to differ significantly in lengths in order to avoid overlap between the final SNaPshot™ products. A difference of 4–6 nucleotides between primer lengths is recommended as a starting point (5–7 nucleotides if running on POP-7™ Polymer).
- The length of a primer can be modified by the addition of non-homologous polynucleotides at the 5′ end. Since the recommended annealing temperature for a SNaPshot™ control primer is 50 degrees C, the melting temperature for the complementary region between any primer and its corresponding template should be at least 50 degrees C.
- Poly (dT), poly (dA), poly (dC), and poly (dGACT) are 5′ non-homologous tails which are predicted to have minimal secondary structures. They have all been used successfully. Generally the signal patterns are not affected by the kinds of tails that are used. The 5′ poly (dT) tails however may interfere with the addition of 3′ ddA.
- The mobility of an oligonucleotide in capillary electrophoresis is determined by its size, nucleotide composition, and dye. Thus the effect of nucleotide composition on mobility can be significant when the primer is short. We strongly recommend that you test primers shorter than 36 nucleotides before being multiplexed to ensure that the final products are spatially resolved when analyzed on the instrument.
- Check primers for possible extendable hairpin structures within each primer and for extendable dimer formation between primers.
- HPLC purification of primers is recommended for oligonucleotides longer than 30 nucleotides. Heterogeneous primer mixtures containing mixed molecular weight oligonucleotides may yield undesired products that will confuse analysis.
For additional suggestions please refer to Appendix A of the SNaPshot™ Multiplex Kit manual.
The SNaPshot™ Multiplex Kit is designed to interrogate up to ten single nucleotide polymorphisms (SNPs) at known locations on one to ten DNA templates in a single tube.
TrueMark MSI Assay
The TrueMark MSI Assay (Cat. No. A45295) is a low-input, single-amplification solution for the rapid identification of microsatellite instability (MSI) in formalin-fixed paraffin-embedded (FFPE) samples from multiple tumor tissue types for research applications. It uses a 6-dye (J6) dye set, the DS-36 Matrix Standard (Dye Set J6) (Cat. No. 4425042) and is compatible with the SeqStudio and 3500 Series genetic analyzers.
Data generated using the TrueMark MSI Assay is meant to be analyzed with TrueMark MSI Analysis Software (downloadable from thermofisher.com/msisoftware). Alternatively, you may use GeneMapper Software (v5.0 or later), but a tumor-normal match must be run and there is no automated calling. We recommend using the full solution for the TrueMark MSI Assay that includes the new TrueMark MSI Assay Analysis Software.
The sample ID markers in the TrueMark MSI Assay are PentaD and TH-01; they are included to detect whether there have been any mistakes in the sample handling process.
We use the following cut-offs based on the paper cited below:
- If >30% of markers show instability, the overall sample is called as MSI-H;
- If <30% of markers show instability, the overall sample is called MSI-L
- If 0% of markers show instability, the overall sample is called MSS
These are the default settings, but you may choose to adjust the percentages.
[Paper citation: Cicek, M et al (2011) Quality Assessment and Correlation of Microsatellite Instability and Immunohistochemical Markers among Population- and Clinic-Based Colorectal Tumors. The Journal of Molecular Diagnostics (13)3.]
The markers for the TrueMark MSI Assay were chosen from literature/publications and bioinformatically determined from GWAS (genome-wide association studies) to be quasi-monomorphic and sensitive across multiple cancer types.
The TrueMark MSI assay was developed with the aim of not requiring a normal control. The sensitivity is comparable to running both a normal sample with the matched tumor sample. In order to detect MSI events at very low abundance (for example, if the pathogenic cells make up only a very small fraction of a biopsy), concurrently running a normal control will solidify the accuracy of the calls and increase the analytical sensitivity of the assay.
The TrueMark MSI Assay has only been tested with FFPE samples at this time.
The GeneScan 600 LIZ dye Size standard v2.0 (Cat. No. 4408399) must be used with the TrueMark MSI Assay Analysis Software, and it has to be ordered separately.
For Research Use Only. Not for use in diagnostic procedures.