PACE (PCR Allelic Competitive Extension) genotyping chemistry is a homogeneous, PCR-based allele-specific technology for the analysis of DNA sequence variants, most commonly SNPs (Single Nucleotide Polymorphisms) and Indels (insertion / deletions).
PACE genotyping chemistry is comprised of two parts:
PACE Genotyping Assay: two allele-specific forward primers and one common, reverse primer. The allele-specific forward primers each have different 3′ terminal bases reflecting the target variant, and a unique 5’ tail sequence which is incorporated as part of the fluorescent signal mechanism.
PACE Genotyping Master Mix: containing all remaining components required for PCR and the generation of fluorescent signals. PACE Genotyping Master Mix contains a novel, universal, fluorescent reporting cassette to produce machine-readable fluorescent signals (FAM and HEX) corresponding to the assay genotypes.
When combined with sample DNA, these components create a PACE Genotyping Reaction, as illustrated in the figure below.
We have extensive knowledge and experience in assay design, especially when it comes to allele-specific PCR. PACE Genotyping Assays are available to purchase either Validated and Unvalidated. Validated assays require customer DNA to validate and optimise, for guaranteed performance. Unvalidated assays are designed in silico and supplied untested.
REQUIRED COMPONENTS
qPCR machine or Thermocycler + Fluorescent plate reader
PCR plate or equivalent and appropriate optically clear seal
Template DNA
PCR-grade water
PACE Genotyping Master Mix or PACE 2.0 Genotyping Master Mix
STEPS TO YOUR PACE GENOTYPING ASSAY DESIGN
Place your order on this page. Our support team will contact you by email.
Fill out an Assay Design Template form with all the information we need to process your custom PACE Genotyping Assay order. We will email you a copy of the template when we first contact you, or your can download a copy here.
Using the information you provide us, we will create your PACE Genotyping Assay designs, order the oligos, and send you design sequences.
Once we receive the oligos, we assemble the assay(s) and then ship an aliquot to you (unvalidated) or test on your DNA samples before shipping the aliquot to you (validated).
Other Products
Saliva DNA Collection and Preservation Devices Dx
Product Info
Document
Product Info
Overview
CE-IVDR marked in accordance with the European Commission Regulation (EU) No. 2017/746.
Ideal for use in in vitro diagnostic workflows
Reliable and cost-effective
Non-invasive, user-friendly sample collection
Sample collection and preservation in one convenient kit
Samples are non-infectious and can be handled and shipped safely
Preserved DNA is stable for 2 years at ambient temperature
High quality DNA is suitable for sensitive downstream applications including PCR, qPCR, sequencing, SNP analysis, microarrays, RFLP and Southern Blot Analysis
Shipping accessories can be purchased separately
Norgen’s Saliva DNA Collection and Preservation Devices are designed for 1) simple and non-invasive saliva collection and 2) preservation of DNA in saliva samples at ambient temperature. Each of the 50 Saliva DNA Collection and Preservation Devices consists of 3 components: (1) Saliva Collection Funnel and Collection Tube, (2) Collection Tube Cap, and (3) Norgen’s Saliva DNA Preservative contained within a sealed squeezable ampoule. Saliva samples are collected by spitting inside the Collection Funnel which has been assembled with the Collection Tube. After collecting the required volume of saliva the Collection Funnel is removed and the contents of the Preservative Ampoule are then added and mixed with the collected saliva. The Saliva Collection Tube is subsequently sent to the laboratory for DNA isolation and analysis. DNA can be isolated from the preserved saliva samples using Norgen’s Saliva DNA Isolation Kit (Cat# Dx45400). Each of Norgen’s Collection Tubes is labeled with a unique serial number that can be used for secure and anonymous tracking of the sample. The saliva DNA in preserved samples is stable for more than 2 years at room temperature. This kit is ideal for collecting and preserving DNA samples for in vitro diagnostic use for medical purposes.
Saliva DNA Preservative
Norgen’s Saliva DNA Preservative is an aqueous storage buffer designed for rapid cellular lysis and subsequent preservation of DNA from fresh specimens. The buffer prevents the growth of Gram-negative and Gram-positive bacteria and fungi, and also inactivates viruses allowing the resulting non-infectious samples to be handled and shipped safely. In addition, the buffer eliminates the need to immediately process or freeze samples and allows the samples to be shipped to centralized testing facilities at ambient temperature. The components of the buffer allow samples to be stored for more than 2 years without any detectable DNA degradation.
DNA Isolation from Preservative
Prior to saliva DNA isolation, vortex the Collection Tube containing preserved saliva for 10 seconds and incubate at 55°C for one hour. Saliva DNA can now be isolated from the preserved saliva samples using Norgen’s spin-column based Saliva DNA Isolation Kit Dx (Cat# Dx45400) or other commercially available methods that have been validated for use with Norgen’s Saliva DNA Collection and Preservation Devices.
NOTE: This product is not available for sale in the United States.
The Collection Device should be kept tightly sealed and stored at room temperature for up to 3 years without any reduction in kit performance (The collection due date is written on the device label).
Once collected, saliva is stable for more than 2 years when kept tightly sealed and stored at room temperature.
The Collection Tube, the Collection Funnel and the Device Container of each Individual Saliva DNA Collection and Preservation Device are recyclable.
Kit Components
Cat. 49000 (50 Devices)
Individual Saliva DNA Collection and Preservation Devices
50
Donor Procedure Flowchart
1
Product Insert
1
Individual Saliva DNA Collection and Preservation Device Contents
AAV Purification from any input – cell fraction or media fraction
High AAV recovery, up to 90%
No specialized equipment needed
Purification from a variety of AAV serotypes (including AAV6 and AAV9)
Yields highly active AAV for in vivo and in vitro experiments
Purification is based on spin column chromatography that uses Norgen’s resin separation matrix
Recombinant adeno-associated virus (AAV) vectors are highly promising tools for both in vitro and in vivo gene transfer. Norgen’s AAV Purification Kits provide fast and simple procedures for concentrating and purifying AAV vectors from cell lysate and cell culture media. Purification is based on precipitation onto Norgen Biotek’s proprietary resin. Contaminating cellular debris is largely removed from the sample via a centrifugation step, while contaminating DNA and RNA is reduced using enzymatic digestion. AAV vector purified in this manner is highly active for use in in vitro and in vivo transduction experiments.
AAV Purification Kit
Norgen’s AAV Purification Kit contains sufficient materials for 15 preparations (33.5 mL per prep of supernatant (SN) or a total of 500 mL of supernatant input). Approximately 1 mL of cell pellet can be purified per prep, up to a maximum of 15 mL of cell pellet in total for the entire kit. Up to 33X sample concentration.
AAV Purification Mini Kit
Each spin column is able to concentrate and purify AAV from 0.5-8 mL of cell pellet, cell culture media, or cells and culture media mixed together. Up to 50X sample concentration. AAV vector purified in this manner is highly active for use in in vitro transduction experiments, and is eluted into a small volume (200 µL). Preparation time for 4 samples is 1.5 hours, with 45 minutes of hands-on time.
AAV Purification Midi Kit
Each spin column is able to concentrate and purify AAV from 8 mL up to 45 mL of input consisting of cell pellet, cell culture media, or cells and culture media mixed together. Up to 50X sample concentration. AAV vector purified in this manner is highly active for use in in vitro transduction experiments, and is eluted into a small volume (1 mL). The kit may be used to purify up to 8 x 25 mL or 4 x 45 mL of samples using the included columns. Preparation time for 4 samples is approximately 2 to 2.5 hours, with 1.5 hours of hands on time.
AAV Purification Maxi Kit (Slurry Format)
Each spin column is able to concentrate and purify AAV from 45 mL to 90 mL of input consisting of cell pellet, cell culture media, or cells and culture media mixed together. Up to 200X sample concentration. AAV vector purified in this manner is highly active for use in in vitro transduction experiments, and is eluted into a small volume (1-10 mL) using the optional concentration step. The kit may be used to purify up to 1 x 900 mL samples or 10 x 45-90 mL samples using the included columns. Preparation time for 1 x 900 mL sample is approximately 2.5 to 3.5 hours, with an optional concentration step requiring an additional 30 min.
At least 5 x 1010 AAV particles as determined by qPCR
AAV Vector Serotype
AAV6, AAV9 and others
Input Type
Cells, media
Input Volume (AAV supernatant)
1 – 33.5 mL SN per prep (500 mL SN in total)
Input Volume (AAV cell pellet)
1 mL cell pellet per prep (15 mL in total)
Time to Complete Purifications
2.5 to 4.5 hours with 1 hour hands on time
In vivo transduction
Yes
Storage Conditions and Product Stability
HL-SAN Nuclease should be stored at -20°C upon arrival. Elution Buffer O should be stored tightly capped at 4°C upon arrival. All other solutions should be kept tightly sealed and stored at room temperature. Once opened, the solutions should be stored at 4°C. This kit is stable for 2 years after the date of shipment.
Component
Cat. 66100 (15 preps)
Cat. 63200 (20 preps)
Cat. 63300 (4-8 preps)
Cat. 63250 (1-10 preps)
Lysis Buffer S
5.5 mL
5.5 mL
5.5 mL
20 mL
DNAse I
–
2 x 25 uL
2 x 25 uL
210 μL
RNAse A
–
60 μL
60 μL
240 μL
HL-SAN Nuclease
102 μL
–
–
–
Binding Buffer A
20 mL
4 mL
4 mL
2 x 8 mL
Purification Solution C
60 mL
–
–
–
Purification Solution D
130 mL
–
–
–
Wash Solution C
2 x 130 mL
60 mL
60 mL
3 x 60 mL
Slurry E
12.5 mL
–
–
2 x 14.5 mL
Elution Buffer O
66 mL
8.5 mL
8.5 mL
66 mL
Protein Neutralizer
4 mL
4 mL
4 mL
4 mL
Spin Columns
–
20
–
–
Mini Spin Columns
–
20
–
–
Midi Spin Columns (grey contents) with Collection Tubes
–
–
8
10
Midi Spin Columns (white contents) with Collection Tubes
–
–
8
–
Maxi Spin Columns (grey contents) with Collection Tubes
–
–
–
10
Maxi Spin Columns (white contents) with Collection Tubes
HiDi® stands for High Discrimination of mismatches at the 3’-terminus of primers in PCR. This myPOLS Biotec enzyme family is optimized for this feature and is the first choice for applications that rely on this property such as allele-specific PCR (asPCR) that is also termed allele-specific amplification (ASA).This polymerase is also available as a full-length Taq DNA polymerase with a nuclease domain, featuring 100% compatibility with hydrolysis probes (TaqMan® probes etc.).Benchmarking with products of competitors conducted by us and others show that the HiDi® DNA polymerase family is the first choice for highly selective PCRs, such as genotyping by allele-specific PCR, HLA genotyping, analysis of single CpG methylation sites or the detection of mutations in a high background of wild-type sequences. By using HiDi® DNA polymerase, less than 10 copies of a mutation can be detected in a background of >10.000 wild-type copies straight away without any other tedious assay optimization.Several independently conducted studies show that HiDi® DNA polymerase is ideally suited for use in asPCR in numerous research areas ranging from mutation detection to genome editing. (read more) For research use and further manufacturing.In case you are aiming to use our RUO products as components or for your development of e.g. an IVD medical device, please contact us.
Casestudies: HiDi® DNA Polymerase: Applications from mutation detection to genome editing (read more)
Example Primer Design
Matching vs. mismatching nucleotide is placed at the 3′-end of the primer for best discrimination results.
Example Results – There´s no accounting for taste
Cilantro: some people love it in their food, some hate it. Here we are detecting a genomic SNP (rs72921001) in HeLa genomic DNA. This SNP is reported to be close to a number of genes coding for olfactory receptors. (Reference: Eriksson N. et al. (2012), “A genetic variant near olfactory receptor genes influences cilantro preference.”)
Considering, that only the C-allele specific primer is extended and yielding in a specific amplicon, we can conclude a genetic predisposition in disliking cilantro, as this SNP is significantly associated with detecting a soapy taste to cilantro.
Allele-specific PCRs were performed from 1 ng/µl of HeLa gDNA in the presence of a realtime dye, indicating the amplification of the C-allele specific primer only. The A-allele specific primer is discriminated, thus not amplified up to 50 cycles.
PCR products were subsequently analysed on a 2.5% agarose gel. Specific product is visualized by ethidium bromide staining at the amplicon length of 109 bp.
Document
HiDi® stands for High Discrimination of mismatches at the 3’-terminus of primers in PCR. This myPOLS Biotec enzyme family is optimized for this feature and is the first choice for applications that rely on this property such as allele-specific PCR (asPCR) that is also termed allele-specific amplification (ASA).
