pcr ruo* reagents - applied bioscienceappliedbioscience.com/docs/asgn/amplidex_flipbook.pdf ·...

877.777.1874 • www.asuragen.com

PCR RUO* Reagents

*For Research Use Only.Not For Use in Diagnostic Procedures.

Fragile X Syndrome and Associated Disorders

• Fragile X Syndrome (FXS) is caused by expansion of CGG repeats in the FMR1 gene, and is the most common known genetic cause of autism.

• Fragile X carriers may be at risk for fragile X-associated tremor/ataxia syndrome (FXTAS), a form of age-related cognitive and motor dysfunction, or primary ovarian insuffi ciency (FXPOI), a cause of premature menopause.

• FXS and other FMR1 disorders impact an estimated 1.5 million individuals in the US, and affect a broad range of populations and ages.

• New therapies and targeted drugs for treating FXS are in development.

The Challenge of Fragile X Molecular Analyses

• Is costly, laborious, and time-consuming (~1 week) to perform

• Requires large amounts of input gDNA (5-10 µg)

• May provide inaccurate CGG repeat quantification

• May have poor sensitivity to detect mosaic alleles

Expansion of CGG repeats and methylation status of the FMR1 gene are used to study FXS and related disorders.

Current FMR1 molecular analyses relies heavily on Southern blot analysis, because laboratory PCR methods have been inadequate. However, Southern blot analysis:

Two Novel PCR Configurations in One Kit

Gene-Specific (GS) FMR1 PCR*

• Full length product peaks for each allele size• All expansions detected to date (>200 full mutations)• More sensitive than Southern blotting• Compatible with multiple sample types

CGG Repeat Primed (RP) FMR1 PCR*

• Full length and CGG peaks for each allele. Expansions are detected irrespective of CGG length

• All expansions detected to date (>200 full mutations)• More sensitive than Southern blotting• Resolves female zygosity• Detects interrupting AGG sequences• Compatible with multiple sample types

Researchers can select the PCR format that best accommodates the needs of their laboratory

Electropherogram of female full mutation Electropherogram of female full mutation

*For Research Use Only. Not For Use in Diagnostic Procedures.

Simple PCR Mastermix Setup & Analysis Workfl ow

CGG RP or Gene-Specifi c PCR

PCR~30 min setup

Capillary Electrophoresis

(CE)~30 min setup

Gene-Specifi c PCR~3.5 hrs

or

CGG RP PCR~5.5 hrs

13 μL PCR mastermix + 2 μL sample (10-40 ng/mL)

CE

PCR

GS PCR Plate orCGG RP PCR Plate

CE Plate

2 μL of PCR product

+ ROX Ladder

Any ABI multi-capillary instrument running

POP-7 liquid polymer

A Comprehensive Product Solution for FMR1 CGG Sizing*


Component Volume Description

Human FMR1 F,R FAM-Primers 50 µLA proprietary FAM-labeled primer pair for human FMR1 gene amplifi cation by PCR

FMR1 CGG Primer 50 µLAn unlabeled CGG primer for probing the number of CGG repeats by PCR amplifi cation

GC-Rich AMP Buffer 1.2 mLA buffered solution containing PCR amplifi cation components optimized for GC-rich DNA

GC-Rich Polymerase Mix 5 µLAn optimized mixture of polymerases for the amplifi cation of GC-rich DNA

Diluent 1 mL A proprietary solution used in PCR mastermix preparation

ROX 1000 Size Ladder 200 µL ROX labeled DNA fragments for sizing from 79 to 1007 bp

Component Volume Description

Human FMR1 F,R FAM-Primers 50 µLA proprietary FAM-labeled primer pair for human FMR1 gene amplifi cation by PCR

FMR1 CGG Primer 50 µLAn unlabeled CGG primer for probing the number of CGG repeats by PCR amplifi cation

GC-Rich AMP Buffer 1.2 mLA buffered solution containing PCR amplifi cation components optimized for GC-rich DNA

GC-Rich Polymerase Mix 5 µLAn optimized mixture of polymerases for the amplifi cation of GC-rich DNA

Diluent 1 mL A proprietary solution used in PCR mastermix preparation

ROX 1000 Size Ladder 200 µL ROX labeled DNA fragments for sizing from 79 to 1007 bp

AmplideX™ protocols, CE instrument modules and GeneMapper confi guration fi les are not part of the research assay.

Validated in Peer-reviewed Publications

Reveals Sensitive and Specific FMR1 Allele Amplicons that Mirror the Results of Southern Blot Analysis?*

Agarose gel image is shown to facilitate comparisons with Southern blot. Capillary electrophoresis is preferred for high resolution FMR1 assessments.


Provides High Resolution CGG Quantification by Capillary Electrophoresis up to 200-250 CGGs*


Detects Full Mutation Alleles up to at least 1300 CGG*


Sizing in red provided from Southern Blot Analysis

Enables Detection to as Low as 1% Full Mutation Allele*


% FM Allele

0%

1%400 pg FM +39.6 ng NOR

3%1.2 ng FM +38.8 ng NOR

5%2 ng FM + 38 ng NOR

Analytical titration of two clinical samples: A male full mutation (FM) allele in a background of a 31 CGG male normal (NOR) allele

High sensitivity detection may enable molecular analysis of unconventional samples, archived materials, or simply those with limited DNA available.

CGG Repeat Primed PCR Resolves Zygosity in Female Samples*

The absence of CGG primed amplicons beyond the size of the detected allele indicates the lack of a longer, heterozygous allele

The presence of CGG primed amplicons beyond the 30 CGG allele indicates the detection of a longer, heterozygous allele


CGG Repeat Primed PCR Detects Interrupting AGG Sequences that can Stabilize the CGG Repeat Region*

The sequence context of AGG interruptions can be determined in many samples

AGG interruptions can be detected in FMR1 alleles with complex genotypes


AmplideX™ FMR1 PCR Summary*


A single PCR reaction provides:

• Reproducible detection of all allele expansions, including low abundance full mutation size mosaics with up to at least 1300 CGG

• Accurate quantifi cation of CGG repeats up to 250 CGG (limited only by CE fragment sizing of full mutations)

• Defi nitive resolution of female homozygous and heterozygous samples

• Detection of interrupting AGG sequences

• Reduction of the need for Southern blot analysis by as much as 10- to 50-fold

AmplideX PCR reagents have been:

• Evaluated in two peer-reviewed publications

• Evaluated at more than two dozen laboratories in the US and abroad

• Used to successfully profi le over 2500 samples, including >200 full mutations with concordance to Southern blot analysis

•

877.777.1874 • www.asuragen.com

Asuragen Inc.2150 Woodward Street, Ste 100

Austin, TX 78744

T: 512-681-5200F: 512-681-5201

pcr ruo* reagents - applied bioscienceappliedbioscience.com/docs/asgn/amplidex_flipbook.pdf ·...

Documents