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qBiomarker Array PreAMP Mix

For loci-specific preamplification prior to real-time PCR to support ultrasensitive cancer mutation detection
  • Nested preamplification improves sensitivity and decreases false positives
  • PCR-based protocol is accessible to most laboratories
  • Integrated, complimentary data analysis simplifies mutation calling
The qBiomarker Array PreAMP Mix enables preamplification of DNA from small amounts of intact or degraded DNA prior to analysis with qBiomarker Somatic Mutation PCR Arrays. The kit contains a mixture of loci-specific primers which, together with the qBiomarker PreAMP PCR Mastermix, uses multiplex PCR preamplification to amplify loci-specific DNA target templates with minimal bias for analysis with qBiomarker Somatic Mutation PCR Arrays or Assays.
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Principle

For successful somatic mutation detection, experimental methods must detect the DNA molecules with mutations of interest in samples that may contain a vast excess of DNA molecules with the wildtype sequence at the same locus. Based off of this principle, there are two factors that affect these experiments: the inherent percentage of mutant DNA in a sample, and the sensitivity of the method. In the case of cancer, a specific mutation may be found in a small percentage of the tumor, which is a characteristic of tumor heterogeneity. Additionally, different types of samples and sample sources affect this ratio of mutant to wildtype DNA as well as the number of DNA molecules that can be assayed. A method’s sensitivity directly reflects how few mutant DNA molecules can be detected within a given amount of wildtype DNA.

Sample types common in cancer research, including formalin-fixed paraffin embedded (FFPE) samples, serum samples, fine needle biopsies, and sorted cells frequently contain only small amounts of amplifiable DNA. Genomic DNA in FFPE samples is fragmented, for example, while DNA in serum samples is heavily diluted. In the case of fine needle biopsies or sorted cells, these samples have small numbers of cells that will yield limited amounts of genomic DNA. Amplifying genomic DNA enables more accurate detection of the mutations in DNA from these sample types.

The qBiomarker PreAMP system uses nested PCR preamplification to improve specificity and decrease false positives. In a 12-cycle PCR reaction, the loci of interest are amplified and are ready to be analyzed with qBiomarker Somatic Mutation PCR Assays and Arrays. The qBiomarker Array PreAMP Mixes contain primers for loci that correspond to the cataloged qBiomarker Somatic Mutation PCR Arrays. Used in combination with the qBiomarker PreAMP Synthesis Kit, this mix produces amplified products specific for the array to be used.

Procedure
DNA is first isolated from FFPE, serum, or small samples using the appropriate QIAamp or DNeasy kit. The qBiomarker PreAMP Synthesis Kit and qBiomarker Array PreAMP Mix are then used to preamplify the DNA using locus-specific primers. qBiomarker Somatic Mutation Arrays are used for mutation detection, and CT values are uploaded into the GeneGlobe Data Analysis tool for data analysis and mutation calls.
Applications

The qBiomarker Array PreAMP Mix is highly suited for preamplification of genomic DNA from samples with low amounts of amplifiable DNA for exclusive use with qBiomarker Somatic Mutation PCR Arrays. Recommended sample types include:

  • FFPE samples
  • Plasma, serum, urine, or other cell-free body fluids
  • Laser capture microdissection samples
  • Fine needle biopsies
  • FACS-sorted cells

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Instrument Technical Documents (1)
For screening disease-focused mutation panels by PCR
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Name qBiomarker PreAMP for Myelodysplastic Syndromes (SMBH-046A)
Species Human (Homo sapiens)
For use with the following mutations:
Gene Symbol ASXL1
RefSeq Accession # ENST00000358956
Nucleotide Change c.1772_1773insA
Amino Acid Change p.Y591fs*1
COSMIC ID 36166
Gene Symbol CBL
RefSeq Accession # NM_005188.1
Nucleotide Change c.1259G>A
Amino Acid Change p.R420Q
COSMIC ID 34077
Gene Symbol CBL
RefSeq Accession # NM_005188.1
Nucleotide Change c.1111T>C
Amino Acid Change p.Y371H
COSMIC ID 34052
Gene Symbol IDH1
RefSeq Accession # NM_005896.2
Nucleotide Change c.395G>T
Amino Acid Change p.R132L
COSMIC ID 28750
Gene Symbol IDH2
RefSeq Accession # NM_002168.2
Nucleotide Change c.515G>T
Amino Acid Change p.R172M
COSMIC ID 33732
Gene Symbol IDH2
RefSeq Accession # NM_002168.2
Nucleotide Change c.516G>T
Amino Acid Change p.R172S
COSMIC ID 34090
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol IDH2
RefSeq Accession # NM_002168
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol CBL
RefSeq Accession # NM_005188
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol IDH1
RefSeq Accession # NM_005896
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol EZH2
RefSeq Accession # NM_004456
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol TET2
RefSeq Accession # NM_001127208
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol ASXL1
RefSeq Accession # ENST00000358956
Nucleotide Change c.2324T>G
Amino Acid Change p.L775*
COSMIC ID 52930
Gene Symbol ASXL1
RefSeq Accession # ENST00000358956
Nucleotide Change c.3202C>T
Amino Acid Change p.R1068*
COSMIC ID 41715
Gene Symbol ASXL1
RefSeq Accession # ENST00000358956
Nucleotide Change c.1888_1909del22
Amino Acid Change p.H630fs*66
COSMIC ID 41716
Gene Symbol ASXL1
RefSeq Accession # ENST00000358956
Nucleotide Change c.2302C>T
Amino Acid Change p.Q768*
COSMIC ID 41717
Gene Symbol CBL
RefSeq Accession # NM_005188.1
Nucleotide Change c.1171G>A
Amino Acid Change p.V391I
COSMIC ID 41272
Gene Symbol DNMT3A
RefSeq Accession # NM_022552.3
Nucleotide Change c.2644C>T
Amino Acid Change p.R882C
COSMIC ID 53042
Gene Symbol EZH2
RefSeq Accession # NM_004456.3
Nucleotide Change c.1936T>A
Amino Acid Change p.Y646N
COSMIC ID 37031
Gene Symbol IDH2
RefSeq Accession # NM_002168.2
Nucleotide Change c.419G>A
Amino Acid Change p.R140Q
COSMIC ID 41590
Gene Symbol IDH2
RefSeq Accession # NM_002168.2
Nucleotide Change c.418C>T
Amino Acid Change p.R140W
COSMIC ID 41877
Gene Symbol TET2
RefSeq Accession # ENST00000380013
Nucleotide Change c.2746C>T
Amino Acid Change p.Q916*
COSMIC ID 43417
Gene Symbol TET2
RefSeq Accession # ENST00000380013
Nucleotide Change c.1648C>T
Amino Acid Change p.R550*
COSMIC ID 41644
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.614A>G
Amino Acid Change p.Y205C
COSMIC ID 43947
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.517G>T
Amino Acid Change p.V173L
COSMIC ID 43559
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.818G>C
Amino Acid Change p.R273P
COSMIC ID 43896
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.713G>T
Amino Acid Change p.C238F
COSMIC ID 43778
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.584T>A
Amino Acid Change p.I195N
COSMIC ID 44877
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.763A>T
Amino Acid Change p.I255F
COSMIC ID 43651
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.536A>T
Amino Acid Change p.H179L
COSMIC ID 43635
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.590T>G
Amino Acid Change p.V197G
COSMIC ID 43905
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.746G>T
Amino Acid Change p.R249M
COSMIC ID 43871
Gene Symbol DNMT3A
RefSeq Accession # NM_022552
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol SF3B1
RefSeq Accession # NM_012433
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol SRSF2
RefSeq Accession # NM_003016
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol U2AF35
RefSeq Accession # NM_006758
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol DNMT3A
RefSeq Accession # NM_022552.3
Nucleotide Change c.2711C>T
Amino Acid Change p.P904L
COSMIC ID 87007
Gene Symbol SF3B1
RefSeq Accession # NM_012433
Nucleotide Change c.000C>G
Amino Acid Change H662Q
COSMIC ID 98000021
Gene Symbol SF3B1
RefSeq Accession # NM_012433
Nucleotide Change c.000A>G
Amino Acid Change K666E
COSMIC ID 98000022
Gene Symbol SF3B1
RefSeq Accession # NM_012433
Nucleotide Change c.000G>T
Amino Acid Change R625L
COSMIC ID 98000027
Gene Symbol SRSF2
RefSeq Accession # NM_003016
Nucleotide Change c.284C>T
Amino Acid Change P95L
COSMIC ID 98000029
Gene Symbol SRSF2
RefSeq Accession # NM_003016
Nucleotide Change c.284C>G
Amino Acid Change P95R
COSMIC ID 98000030
Gene Symbol U2AF35
RefSeq Accession # NM_006758
Nucleotide Change c.470A>G
Amino Acid Change Q157R
COSMIC ID 98000032
Gene Symbol U2AF35
RefSeq Accession # NM_006758
Nucleotide Change c.101C>A
Amino Acid Change S34Y
COSMIC ID 98000034
Gene Symbol NRAS
RefSeq Accession # NM_002524
Nucleotide Change c.183A>T
Amino Acid Change p.Q61H
COSMIC ID 585
Gene Symbol NRAS
RefSeq Accession # NM_002524
Nucleotide Change c.38G>A
Amino Acid Change p.G13D
COSMIC ID 573
Gene Symbol NRAS
RefSeq Accession # NM_002524
Nucleotide Change c.183A>C
Amino Acid Change p.Q61H
COSMIC ID 586
Gene Symbol NRAS
RefSeq Accession # NM_002524
Nucleotide Change c.52G>A
Amino Acid Change p.A18T
COSMIC ID 577
Gene Symbol RUNX1
RefSeq Accession # ENST00000300305
Nucleotide Change c.167T>C
Amino Acid Change p.L56S
COSMIC ID 24756
Gene Symbol RUNX1
RefSeq Accession # ENST00000300305
Nucleotide Change c.496C>T
Amino Acid Change p.R166*
COSMIC ID 24769
Gene Symbol RUNX1
RefSeq Accession # ENST00000300305
Nucleotide Change c.610C>T
Amino Acid Change p.R204*
COSMIC ID 25124
Gene Symbol RUNX1
RefSeq Accession # ENST00000300305
Nucleotide Change c.223G>T
Amino Acid Change p.D75Y
COSMIC ID 26150
Gene Symbol RUNX1
RefSeq Accession # ENST00000300305
Nucleotide Change c.593A>T
Amino Acid Change p.D198V
COSMIC ID 36059
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.818G>A
Amino Acid Change p.R273H
COSMIC ID 10660
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.832C>T
Amino Acid Change p.P278S
COSMIC ID 10939
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.742C>T
Amino Acid Change p.R248W
COSMIC ID 10656
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.844C>T
Amino Acid Change p.R282W
COSMIC ID 10704
Gene Symbol TP53
RefSeq Accession # NM_000546
Nucleotide Change c.797G>T
Amino Acid Change p.G266V
COSMIC ID 10958
Gene Symbol NRAS
RefSeq Accession # NM_002524
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol ASXL1
RefSeq Accession # NM_015338
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol RUNX1
RefSeq Accession # NM_001754
Nucleotide Change copy number
Amino Acid Change copy number