Amplifluor® Technology

Amplifluor® Universal Systems are based on energy transfer from an excited fluorophore to a complexed acceptor moiety that results in quenching of the fluorescence.  This quenching is accomplished by linking the fluorophore and the acceptor 4-(dimethylamino)azo benzene sulfonic acid (DABSYL) to an oligonucleotide primer.  The optimised design of the UniPrimers™ results in a large increase in fluorescent emission only upon incorporation into the amplification products produced during each PCR cycle.  Unincorporated primers have low fluorescence, eliminating the need to purify the PCR reaction products prior to quantitation.

Each UniPrimer™consists of a different 3' primer sequence and a 5' hairpin region that is labelled with a unique energy transfer pair.  The primer sequences enable the UniPrimers™ to work with target specific unlabelled primers to the researcher's target specific sequence.  The target specific primers are synthesised with a 5' tail sequence identical to the 3' region of a particular Uniprimer™  , allowing it to hybridise to the PCR product.

During PCR, the incorporated UniPrimer™ serves as a template for DNA polymerisation.  This process involves the displacement and replication of the hairpin sequence catalysed by DNA polymerase.  The fluorophore is located at the 5' base of the primer and the quencher is linked to the nucleotide that is complementary to the 5' base.  Efficient fluorescence quenching occurs when the primer is in this hairpin conformation.  Upon incorporation into the doublestranded product, the hairpin structure unfolds and disrupts the energy-transfer between the fluorophore and the quencher.  The fluorescent signal produced during each PCR cycle directly correlates to the amount of amplified DNA generated. 
 

Amplifluor Universal Detection System has two parts.  The first step incorporates a hybridisation sequence for the reporter UniPrimer, which is used in the second step.  During DNA polymerisation, the UniPrimer hairpin structure is displaced, generating a fluorescent signal that correlates directly to the amount of amplification product.

 Amplifluor® SNPs Genotyping System

The Amplifluor® SNPs Genotyping System for Assay Development is designed for new users unfamiliar with the technology.  This kit contains the fluorescein-, sulforhodamine- and JOE-labelled Amplifluor™ UniPrimers™ as well as control primers and templates.

Principle: 

The Amplifluor®  SNPs Genotyping System is a single tube system utilising two Amplifluor®  SNPs Primers and three unlabelled primers.  The pair of Amplifluor®  SNPs Primers are labelled either with fluorescein (FAM) and sulforhodamine (SR) or with FAM and a 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE).  A single common reverse primer and two SNP specific primers are designed to amplify across the SNP, each with a 5' tail that corresponds to one of the two Amplifluor®  SNPs Primers.  As SNP-specific PCR products are generated, the primer sequence of the Amplifluor®  SNPs Primers hybridises to the complement of the tail sequence on the newly generated PCR product and, in conjunction with the common reverse primer, amplifies the product further.  Depending on which base is present in the target sequence, a FAM or a JOE signal is generated.  (When the Amplifluor®  SNPs Primers are labelled with FAM and SR are used, the signal produced is either FAM or SR).  Heterozygotes result in signals from both fluorophores.  The Amplifluor®  SNPs Primers and the common reverse primer are used in a 10-fold excess over the SNP specific tailed primers to ensure high yield of the fluorescent product.  The same Amplifluor SNPs Primers can be used for scanning many different SNPs since the discrimination of each SNP only requires the user to have three unmodified oligonucleotides synthesised.

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 Amplifluor® SNPs HT Genotyping System FAM-JOE

Description
The Amplifluor™ SNPs HT Genotyping System FAM-JOE is designed for users familiar with Amplifluor™ and is for users of real-time PCR detection instruments.

Tested Applications: Software needed

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 Amplifluor® SNPs HT Genotyping System FAM-SR

Description
The Amplifluor™ SNPs HT Genotyping System FAM-SR is designed for users familiar with Amplifluor™ and is for use with fluorescent plate readers.

Tested Applications: Software needed

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 Amplifluor® Assay Optimization Buffers

The buffers and PCR addititive are provided for use with the Amplifluor® SNPs Genotyping System for Assay Development (Cat No. S7907) in assay optimization. The S buffers are meant for use with the Amplifluor® FAM and SR Primer pair and in a fluorescent plate reader; the S Plus buffers contain a passive reference dye and are meant for use with the Amplifluor® FAM and JOE Primer pair and in a real-time PCR instrument such as the ABI PRISM®. Use of these buffers is recommended when use of the standard buffer provided in S7907 fail to generate clear clusters. Because the GC percentage can vary between the allele-specific and reverse primers, it is recommended that all the appropriate optimization buffers be tested with a small number of samples. In general, if the region flanking the SNP has a low GC content (high AT content), increasing the MgCl2 concentration may improve the results. Conversely, if the region flanking the SNP has a high GC content, decreasing the MgCl2 concentration may improve the results. The PCR Additive is recommended for SNPs with extremely high GC content. The optimal working concentration for the PCR Additive must be determined by end user. It is most often used at a 0.125 M, 0.25 M or 0.5 M final concentration.

ABI PRISM® is a registered trademark of Applied Biosystems, Inc

Components:

10X Reaction Buffer S (10mM MgCl2), 100μl
10X Reaction Buffer S (15mM MgCl2), 100μl
10X Reaction Buffer S (25mM MgCl2), 100μl
10X Reaction Buffer S plus (10mM MgCl2), 100μl
10X Reaction Buffer S plus (15mM MgCl2), 100μl
10X Reaction Buffer S plus (25mM MgCl2), 100μl
GC Rich PCR Additive (5.0M), 100μl

Storage/Handling:
Store at -20°C. Some material might be light sensitive. Refer to product label.

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 Amplifluor® FAM-JOE Assay Optimization Buffers

Presentation:
Liquid

10X Reaction Buffer S plus (10mM MgCl2), 5ml
10X Reaction Buffer S plus (15mM MgCl2), 5ml
10X Reaction Buffer S plus (25mM MgCl2), 5ml
GC Rich PCR Additive (5.0M), 5ml

Storage/Handling:
Store at -20°C. Material is light sensitive avoid exposure.

Application Notes:
The buffers and PCR addititive are provided for use with the Amplifluor® SNPs HT Genotyping System, FAM-JOE (Cat. No. S7909) in assay optimization. The S Plus buffers contain a passive reference dye are meant for use with the Amplifluor® FAM and JOE Primer pair and in a real-time PCR instrument such as the ABI PRISM®. Use of these buffers is recommended when use of the standard buffers provided in S7909 fail to generate clear clusters. Because the GC percentage can vary between the allele-specific and reverse primers, it is recommended that all the appropriate optimization buffers be tested with a small number of samples. In general, if the region flanking the SNP has a low GC content (high AT content), increasing the MgCl2 concentration may improve the results. Conversely, if the region flanking the SNP has a high GC content, decreasing the MgCl2 concentration may improve the results. The PCR Additive is recommended for SNPs with extremely high GC content. The optimal working concentration for the PCR Additive must be determined by end user. It is most often used at 0.125 M, 0.25 M and 0.5 M final concentration.

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 Amplifluor® FAM-SR Assay Optimization Buffers

Presentation:
Liquid

10X Reaction Buffer S (10mM MgCl2), 5ml
10X Reaction Buffer S (15mM MgCl2), 5ml
10X Reaction Buffer S (25mM MgCl2), 5ml
GC Rich PCR Additive, 5ml

Storage/Handling: Store at -20ºC

Application Notes:
For use with S7908 Amplifluor® SNPs HT Genotyping System FAM-SR in assay optimization

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 Amplifluor® UniPrimer™ II Fluorescein

The Amplifluor™ UniPrimer™-fluorescein is a hairpin primer labeled with a fluorescent energy transfer pair (fluorescein and non-fluorescent quencher) and a 3' single-stranded oligonucleotide tail and is provided at a concentration of 5.0 μM.
Fluorescence (Linear:Hairpin): 39. Excitation = 495nm; Emission = 516nm.

Storage/Handling:
Minimize exposure to light as this material is light sensitive. Make sterile aliquots of bulk solution. Store long term at -65°C to -75°C. Packaged product for end user may be stored in the dark at -15°C to -25°C.

Recommended diluent: autoclaved, reagent grade, deionized water only.

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