PACE® Multiplex Master Mix (Endpoint) is a 2x PCR master mix for running two SNP or Indel genotyping assays in one reaction well, or for detecting a reference sequence plus up to three sequences of interest. This user guide covers reagent preparation, assay assembly for genotyping and sequence detection, reaction volumes for 96-, 384- and 1536-well formats, thermal cycling, four-colour fluorescence reading and data interpretation.
Looking for the trial kit instructions instead? See the PACE® Multiplex Master Mix Trial Kit user guide.
PACE Multiplex at a glance
- Applications: two SNP or Indel genotyping assays in one well, or detection of a reference sequence and up to three targets of interest
- Fluorophores: FAM, HEX, ATTO 590 and ATTO 647N, with ATTO 680 as passive reference dye
- Pack sizes: 2.5 mL to 1,000 mL (500 to 200,000 reactions at 10 µL)
- DNA input: 1-10 ng gDNA per well, purified or crudely extracted
- Thermal cycling: 15 min activation at 94 °C, 10 touchdown cycles (65-57 °C), 30 cycles at 57 °C
- Storage: -20 °C / -80 °C for two years, or 4 °C for two weeks protected from light
1. PRODUCT DETAILS
2. DESCRIPTION
PACE Multiplex (PCR Allelic Competitive Extension) genotyping chemistry is a homogeneous, PCR-based allele-specific technology for the simultaneous analysis of two Single Nucleotide Polymorphisms (SNPs) or insertion/deletions (Indels), or detection of sequences of interest.
The PACE Multiplex chemistry is comprised of two parts:
- PACE Genotyping Assay: comprising two pairs of allele-specific forward primers and two common, reverse primers for SNPs or Indels, or four pairs of primers for detecting genes of interest.
- PACE Multiplex Master Mix: containing all components required for PCR and generation of fluorescent signals.
When combined and mixed with template DNA, these components create a PACE Multiplex Genotyping Reaction.
3. STORAGE AND SHELF LIFE
PACE Multiplex Master Mix is shipped on blue ice. Upon arrival, store at -20˚C/-80˚C (stable for two years); multiple freeze/thaw cycles are not recommended. PACE Multiplex Master Mix can be aliquoted into light-protective tubes to reduce the need for repeated freeze-thaw cycles. The mix can also be stored at 4˚C for two weeks (protected from light).
4. SAFETY WARNINGS AND PRECAUTIONS
This product should only be handled by trained laboratory personnel. It is advisable to wear suitable personal protective equipment (PPE) when using the product. In case of contact with skin or eyes, wash immediately with water.
5. KIT COMPONENTS
PACE Multiplex Master Mix (supplied at 2x concentration), containing a specifically engineered Taq polymerase, universal fluorescent reporting cassettes, dNTPs, buffer, performance enhancers, MgCl2 at 4.4 mM (2.2 mM at 1x concentration) and a passive reference dye, ATTO 680.
REQUIRED COMPONENTS
- Fluorescent plate reader or qPCR machine capable of reading the fluorophores in Table 1
- PCR plate or equivalent and appropriate optically clear seal
- Template DNA
- PCR-grade water
- Genotyping assays (see section 8).
| FLUOROPHORE | EXCITATION (nm) | EMISSION (nm) |
| FAM | 485 | 520 |
| HEX | 520 | 560 |
| ATTO 590 | 590 | 620 |
| ATTO 647N | 649 | 662 |
| ATTO 680 Reference Dye | 681 | 698 |
6. REFERENCE DYE
PACE Multiplex Master Mix is supplied with reference dye ATTO 680. Please check your instrument for reference dye compatibility; should you require further assistance, please contact the manufacturer of your qPCR instrument or plate reader or contact 3CR Bioscience’s Technical Support team.
7. MECHANISM OF ACTION
PACE Multiplex Master Mix uses a novel, universal, fluorescent reporting cassette to produce machine-readable fluorescent signal. The master mix contains four universal quenched fluorescent reporting cassettes, labelled with the fluorophores FAM, HEX, ATTO 590 and ATTO 647N.
When used for genotyping applications, two genotyping assays are used, each comprised of two competitive allele-specific forward primers (which differ in their terminal 3’ bases and unique 5’ tail sequences), and a common reverse primer. One multiplex assay reports with FAM and HEX, whilst the other reports with ATTO 590 and ATTO 647N.
When PCR is initiated, the allele-specific primers bind with their 3’ ends at the SNP of interest. Both allele-specific primers will bind if the SNP is heterozygous, whereas only one or other of the primers will bind if the SNP is homozygous. At the same time, the common reverse primer will bind on the opposite strand.
As PCR proceeds, the tail sequences of allele-specific forward primers become incorporated into the amplicon and the corresponding tail sequence complement is generated. At this point the quenched, fluorescent reporting cassettes bind to their appropriate tail sequence complements, becoming unquenched and producing a light signal. If the genotype of the SNP is homozygous, only one of the possible fluorescent signals will be generated, whereas if the SNP is heterozygous, the result will be a mixed fluorescent signal.
Alternatively, if PACE Multiplex Master Mix is used for sequence detection applications, one forward primer (containing a 5’ tail sequence, as for the allele-specific primers in the genotyping application above) can be designed to a reference sequence whilst the other three forward primers can be designed to individual targets of interest, with a reverse primer designed for each PCR. When testing for the presence or absence of sequences, the forward and reverse primers will bind, and the PCR will proceed as described above for a SNP.
The reference gene will always be present, so the assay will produce a homozygous fluorescent signal for the fluorophore selected for the reference sequence assay; this shows that the reaction is functioning as expected. The other assay primers will bind to their target sequences of interest (if present) and the appropriate signal(s) will be produced. If a gene of interest is present, both signals will be generated, resulting in a heterozygous sample.
8. MULTIPLEX ASSAYS
PACE Genotyping Assay designs are available free-of-charge by using our complimentary PACE Assay design service at www.3crbio.com or by contacting support@3crbio.com
A. GENOTYPING ASSAYS
PACE Multiplex Master Mix allows the use of two PACE Genotyping Assays simultaneously in one reaction well. Tables 2a and 2b show how to assemble assay mixes for PACE Genotyping Assays 1 and 2 from the constituent primers.
Pre-existing KASP™ and Amplifluor® assays (i.e., which contain allele-specific primers with FAM/HEX reporting tails) can also be used in conjunction with the PACE Multiplex genotyping chemistry. If used, these assays would replace those described in Table 2a.
PACE GENOTYPING ASSAY 1
| PRIMER | FINAL CONCENTRATION (µM) | VOL. REQUIRED FOR 100 µL ASSAY MIX (µL) |
| Assay 1 allele-specific forward primer 1 – FAM sequence tail (100 µM) | 12 | 12 |
| Assay 1 allele-specific forward primer 2 – HEX sequence tail (100 µM) | 12 | 12 |
| Assay 1 common, reverse primer (100 µM) | 30 | 30 |
| PCR-grade water | – | 46 |
| TOTAL | – | 100 |
PACE GENOTYPING ASSAY 2
| PRIMER | FINAL CONCENTRATION (µM) | VOL. REQUIRED FOR 100 µL ASSAY MIX (µL) |
| Assay 2 allele-specific forward primer 1 – ATTO 590 sequence tail (100 µM) | 12 | 12 |
| Assay 2 allele-specific forward primer 2 – ATTO 647N sequence tail (100 µM) | 12 | 12 |
| Assay 2 common, reverse primer (100 µM) | 30 | 30 |
| PCR-grade water | – | 46 |
| TOTAL | – | 100 |
B. SEQUENCE DETECTION ASSAYS
Table 3 shows how to assemble a PACE Genotyping Assay mix to detect multiple targets of interest.
9. DNA QUALITY AND QUANTITY
It is recommended to use 1-10 ng of gDNA per reaction well, though this will vary with organism genome size (large genomes will require a proportionately larger DNA mass). For optimal results, purified and well-normalised DNA samples should be used. However, when using PACE Multiplex Master Mix in high throughput, purified DNA is often not commercially practical. DNA that has been crudely extracted works well with PACE Multiplex Master Mix, but such samples should be tested before commencing large-scale work. Empirical optimisation of DNA concentration by testing a sample dilution range test is the most sensible approach. If DNA samples contain EDTA, the concentration at the final reaction concentration should be no higher than 0.1 mM.
10. CONTROLS
To improve confidence in the genotyping data, control samples should be used on the PCR plate in addition to the test samples. Negative controls (no-template controls, or NTCs) should always be used, and consist of the same buffer used to hydrate the DNA samples, dispensed into several wells of the PCR plate. Positive controls can also be used, if available, and should consist of DNA samples of known genotype.
When viewing the genotyping data, NTCs should show no amplification and remain around the origin of the cluster plot (see Figure 1), giving confidence that any amplification observed is real. Any amplification observed in the NTC wells would indicate contamination or non-specific amplification. The positive control samples should cluster in the expected regions for their genotype.
11. REACTION PROCEDURE
A. ARRAYING TEMPLATE DNA
A liquid-handling system appropriate to the number of DNA samples to array should be used. DNA samples can also be arrayed manually if working with a small number of samples.
PACE Multiplex Master Mix can be used with hydrated (Table 4a) or dry (Table 4b) DNA samples. Both approaches work equally well but have practical advantages and disadvantages. If low numbers of samples are to be genotyped, it is not worth drying the DNA samples. However, if high numbers of samples are to be genotyped in one run, drying the DNA samples can improve the resulting genotyping data. Hydrated DNA arrayed in a PCR plate will quickly begin to evaporate differentially across the plate (samples near the edges evaporate more quickly than those in the middle). Variation in DNA volumes across the plate will lead to variation in the final reaction concentrations, causing sub-optimal genotyping results. When drying the DNA into the plate wells the user must ensure that the DNA has been dried to completion. Dried DNA samples will be stable long term at ambient temperature. If a very small reaction volume is to be used (for example 1.0 µL total volume), it might not be possible to accurately dispense 0.5 µL of DNA and 0.5 µL of total reaction mix. In this example, drying the sample would allow a more realistic 1.0 µL of total reaction mix to be dispensed to the well. To dry the DNA, once dispensed into a PCR plate, the plate should be centrifuged to ensure the samples are in the bottom of the wells and placed in a laboratory fan oven for one hour at around 55˚C, or until the samples have visibly dried. When assembling the total reaction mix, water must be added in the correct proportion to account for the missing volume of the DNA template. See Tables 4a and 4b for details.
B. REACTION ASSEMBLY
PACE Multiplex Master Mix can be used with any reaction plate or well volume; it is not necessary to use a different product depending on reaction volume. It is very important that PACE Multiplex Master Mix is used at a final 1x concentration. However, the exception to this is in the PACE Genotyping Assay volumes shown in Tables 4a & 4b – the PACE Genotyping Assay volume is ignored as it does not cause any functionally meaningful dilution and does not affect the performance.
If using PACE Multiplex Master Mix for sequence detection applications, just one PACE Genotyping Assay mix would be used so the ‘PACE Assay 2’ in Tables 4a & 4b would be ignored.
C. PACE MULTIPLEX GENOTYPING REACTION DISPENSING & PLATE SEALING
The total reaction mix must now be dispensed into the PCR plate wells. As with DNA dispensing, use a liquid handling system that is appropriate to the scale of the work. Once the total reaction mix has been dispensed, the PCR plate must be sealed with an optically clear seal and centrifuged to ensure all components are at the bottom of the wells.
D. THERMAL CYCLING
PACE Multiplex Genotyping Reactions should be thermally cycled according to the protocol in Table 5a.
E. FLUORESCENT SIGNAL DETECTION
After thermal cycling is complete, the fluorescent signal data should be collected using an appropriate fluorescent plate reader or qPCR machine in endpoint mode. Table 6 details the optimal suggested wavelength and bandwidth settings for each filter to minimise crosstalk between the different filters.
| FLUOROPHORE | EXCITATION (nm) | EMISSION (nm) |
| FAM | 485 (10 nm) | 520 (10 nm) |
| HEX | 520 (10 nm) | 560 (10 nm) |
| ATTO 590 | 590 (10 nm) | 620 (10 nm) |
| ATTO 647N | 635 (10 nm) | 670 (10 nm) |
| ATTO 680 | 675 (10 nm) | 705 (10 nm) |
If the genotype clusters are not sufficiently defined after running the initial thermal cycling protocol, the plate should be cycled for an additional three cycles (see Table 5b) then the fluorescent signal data collected again. The additional cycling/data analysis can be repeated until tight and well-separated clusters are observed; however, it is recommended that this step is repeated a maximum of four times.
It is important that the fluorescent signal is read at or below 40˚C. If using a qPCR instrument, an additional temperature-controlled reading step should be included after the final PCR step or used separately to it. The temperature-controlled reading step should be used with both the main PCR (as described in Table 5a) and with any subsequent additional cycling steps (Table 5b). If using a fluorescent plate reader, the addition of this temperature-controlled reading step to the thermal cycling protocol should not be necessary as the PCR plate will have cooled sufficiently by the plate-reading stage.
F. INTERPRETATION OF DATA
The HEX, FAM, ATTO 590, and ATTO 647N fluorescence signal data produced by PACE Multiplex Genotyping Reactions should be analysed and interpreted as a cluster plot using cluster analysis software or with Microsoft Excel (see Figure 1).
ROX passive reference dye can also be used to eliminate the effect of well-to-well liquid volume differences from the resulting cluster plot data. The inclusion of a passive reference leads to tighter clustering and, as a result, more accurate scoring of data.
12. ORDERING INFORMATION
For ordering details, please visit www.3crbio.com.
13. SUPPORT
If you require any support with the use of PACE Multiplex Master Mix or other 3CR Bioscience products, please contact our Technical Support team on support@3crbio.com.
14. LEGAL INFORMATION
For Research Use Only. Not for use in diagnostic procedures.
3CR Bioscience disclaims all warranties with respect to this documentation.
The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly or by implication. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser’s activities for a fee or other form of consideration.
KASP™ is a trademark of LGC Biosearch Technologies
Amplifluor® is a registered trademark of Merck KGaA
©2024 3CR Bioscience Ltd. All rights reserved. Intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
