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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 Thyroid Cancer (SMBH-028A)
Species Human (Homo sapiens)
For use with the following mutations:
Gene Symbol BRAF
RefSeq Accession # NM_004333
Nucleotide Change c.1803A>T
Amino Acid Change p.K601N
COSMIC ID 6265
Gene Symbol BRAF
RefSeq Accession # NM_004333
Nucleotide Change c.1789C>G
Amino Acid Change p.L597V
COSMIC ID 470
Gene Symbol CDKN2A
RefSeq Accession # NM_000077
Nucleotide Change c.237_238CC>TT
Amino Acid Change p.R80*
COSMIC ID 13524
Gene Symbol CTNNB1
RefSeq Accession # NM_001904
Nucleotide Change c.110C>A
Amino Acid Change p.S37Y
COSMIC ID 5666
Gene Symbol CTNNB1
RefSeq Accession # NM_001904
Nucleotide Change c.119C>T
Amino Acid Change p.T40I
COSMIC ID 5708
Gene Symbol CTNNB1
RefSeq Accession # NM_001904
Nucleotide Change c.146A>G
Amino Acid Change p.K49R
COSMIC ID 5709
Gene Symbol CTNNB1
RefSeq Accession # NM_001904
Nucleotide Change c.130C>T
Amino Acid Change p.P44S
COSMIC ID 5704
Gene Symbol EGFR
RefSeq Accession # NM_005228
Nucleotide Change c.2497T>G
Amino Acid Change p.L833V
COSMIC ID 13424
Gene Symbol EGFR
RefSeq Accession # NM_005228
Nucleotide Change c.2193G>A
Amino Acid Change p.W731*
COSMIC ID 13432
Gene Symbol EGFR
RefSeq Accession # NM_005228
Nucleotide Change c.2572C>A
Amino Acid Change p.L858M
COSMIC ID 12366
Gene Symbol EGFR
RefSeq Accession # NM_005228
Nucleotide Change c.2239_2258>CA
Amino Acid Change p.L747_P753>Q
COSMIC ID 12387
Gene Symbol GNAS
RefSeq Accession # NM_000516.3
Nucleotide Change c.681G>T
Amino Acid Change p.Q227H
COSMIC ID 27900
Gene Symbol GNAS
RefSeq Accession # NM_000516.3
Nucleotide Change c.601C>A
Amino Acid Change p.R201S
COSMIC ID 27899
Gene Symbol HRAS
RefSeq Accession # NM_005343
Nucleotide Change c.37G>T
Amino Acid Change p.G13C
COSMIC ID 488
Gene Symbol HRAS
RefSeq Accession # NM_005343
Nucleotide Change c.183G>C
Amino Acid Change p.Q61H
COSMIC ID 503
Gene Symbol KRAS
RefSeq Accession # NM_004985
Nucleotide Change c.35G>A
Amino Acid Change p.G12D
COSMIC ID 521
Gene Symbol KRAS
RefSeq Accession # NM_004985
Nucleotide Change c.182A>T
Amino Acid Change p.Q61L
COSMIC ID 553
Gene Symbol KRAS
RefSeq Accession # NM_004985
Nucleotide Change c.57G>C
Amino Acid Change p.L19F
COSMIC ID 12703
Gene Symbol KRAS
RefSeq Accession # NM_004985
Nucleotide Change c.38_39GC>AT
Amino Acid Change p.G13D
COSMIC ID 531
Gene Symbol TSHR
RefSeq Accession # NM_000369
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol GNAS
RefSeq Accession # NM_000516
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol CTNNB1
RefSeq Accession # NM_001904
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol RET
RefSeq Accession # NM_020975
Nucleotide Change copy number
Amino Acid Change copy number
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 PIK3CA
RefSeq Accession # NM_006218.1
Nucleotide Change c.1564G>A
Amino Acid Change p.E522K
COSMIC ID 28540
Gene Symbol PIK3CA
RefSeq Accession # NM_006218.1
Nucleotide Change c.1637A>T
Amino Acid Change p.Q546L
COSMIC ID 25041
Gene Symbol PIK3CA
RefSeq Accession # NM_006218.1
Nucleotide Change c.3001C>A
Amino Acid Change p.L1001I
COSMIC ID 29095
Gene Symbol PIK3CA
RefSeq Accession # NM_006218.1
Nucleotide Change c.3184A>G
Amino Acid Change p.I1062V
COSMIC ID 29108
Gene Symbol PIK3CA
RefSeq Accession # NM_006218.1
Nucleotide Change c.3073A>G
Amino Acid Change p.T1025A
COSMIC ID 771
Gene Symbol PIK3CA
RefSeq Accession # NM_006218.1
Nucleotide Change c.1624G>C
Amino Acid Change p.E542Q
COSMIC ID 17442
Gene Symbol PIK3CA
RefSeq Accession # NM_006218.1
Nucleotide Change c.3074C>T
Amino Acid Change p.T1025I
COSMIC ID 36285
Gene Symbol RET
RefSeq Accession # NM_020975
Nucleotide Change c.2753T>C
Amino Acid Change p.M918T
COSMIC ID 965
Gene Symbol RET
RefSeq Accession # NM_020975
Nucleotide Change c.2647_2648GC>TT
Amino Acid Change p.A883F
COSMIC ID 977
Gene Symbol RET
RefSeq Accession # NM_020975
Nucleotide Change c.2304G>C
Amino Acid Change p.E768D
COSMIC ID 21338
Gene Symbol RET
RefSeq Accession # NM_020975
Nucleotide Change c.1894_1906>AGCT
Amino Acid Change p.E632_T636>SS
COSMIC ID 1048
Gene Symbol TSHR
RefSeq Accession # NM_000369.1
Nucleotide Change c.1868C>T
Amino Acid Change p.A623V
COSMIC ID 26417
Gene Symbol TSHR
RefSeq Accession # NM_000369.1
Nucleotide Change c.1887G>T
Amino Acid Change p.L629F
COSMIC ID 26422
Gene Symbol TSHR
RefSeq Accession # NM_000369.1
Nucleotide Change c.1535T>A
Amino Acid Change p.L512Q
COSMIC ID 26448
Gene Symbol TSHR
RefSeq Accession # NM_000369.1
Nucleotide Change c.1358T>C
Amino Acid Change p.M453T
COSMIC ID 26449
Gene Symbol TSHR
RefSeq Accession # NM_000369.1
Nucleotide Change c.1456A>T
Amino Acid Change p.I486F
COSMIC ID 26450
Gene Symbol TSHR
RefSeq Accession # NM_000369.1
Nucleotide Change c.1703T>C
Amino Acid Change p.I568T
COSMIC ID 26451
Gene Symbol TSHR
RefSeq Accession # NM_000369.1
Nucleotide Change c.1887G>C
Amino Acid Change p.L629F
COSMIC ID 26453
Gene Symbol TSHR
RefSeq Accession # NM_000369.1
Nucleotide Change c.1899C>A
Amino Acid Change p.D633E
COSMIC ID 26477
Gene Symbol TSHR
RefSeq Accession # NM_000369.1
Nucleotide Change c.1535T>G
Amino Acid Change p.L512R
COSMIC ID 26484
Gene Symbol AKT1
RefSeq Accession # ENST00000349310
Nucleotide Change c.49G>A
Amino Acid Change p.E17K
COSMIC ID 33765
Gene Symbol AKT1
RefSeq Accession # NM_005163.2
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol BRAF
RefSeq Accession # NM_004333
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol EGFR
RefSeq Accession # NM_005228
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol CDKN2A
RefSeq Accession # NM_000077
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol MET
RefSeq Accession # NM_000245
Nucleotide Change c.3029C>T
Amino Acid Change p.T1010I
COSMIC ID 707
Gene Symbol KRAS
RefSeq Accession # NM_004985
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol HRAS
RefSeq Accession # NM_005343
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol NRAS
RefSeq Accession # NM_002524
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol PIK3CA
RefSeq Accession # NM_006218.1
Nucleotide Change copy number
Amino Acid Change copy number
Gene Symbol MET
RefSeq Accession # NM_000245
Nucleotide Change copy number
Amino Acid Change copy number