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Do not freeze reagents.
B. Store MGAC, PIK3CA MMX-1, PIK3CA MMX-2, PIK3CA MMX-3, PIK3CA MC, and DNA SD at 2ºC to 8°C. Once opened, these reagents are stable for 4 uses within 90 days or until the expiration date, whichever comes first.
C. PIK3CA MMX-1, PIK3CA MMX-2, PIK3CA MMX-3, and working MMX (prepared by the addition of MGAC to PIK3CA MMX-1 or PIK3CA MMX-2 or PIK3CA MMX-3) should be protected from prolonged exposure to light.
D. Store working MMX at room temperature in the dark. The prepared samples and controls must be added within 1 hour of preparation of the working MMX.
E. Processed specimens (extracted DNA) are stable for up to 24 hours at 15ºC to 30°C, up to 14 days at 2ºC to 8°C, up to 60 days at -15°C to -25°C and after undergoing 3 freeze thaws when stored at -15°C to -25°C. Extracted DNA should be amplified within the recommended storage periods or before the expiration date of the cobas^® DNA Sample Preparation Kit used to extract the DNA, whichever comes first.
F. Amplification must be started within 1 hour from the time that the processed samples and controls are added to the working MMX (prepared by the addition of MGAC to PIK3CA MMX-1 or PIK3CA MMX-2 or PIK3CA MMX-3).", "Language": "en", "Country": "XG", "Code": "Storage Conditions (Product)" }, { "Name": "Assay Time", "Value": "The sample extraction process takes about 3 hours. This may vary according to the number of samples being extracted.

The Amplification and Detection step takes 90 minutes.", "Language": "en", "Country": "XG", "Code": "Assay Time" }, { "Name": "Principle", "Value": "The cobas^® PIK3CA Mutation Test (cobas PIK3CA Test) is a research use only (RUO) real-time PCR test for the qualitative detection and identification of mutations in exons 1, 4, 7, 9, and 20 of the phosphoinositide-3-kinase, catalytic, alpha (PIK3CA) gene in DNA derived from formalin-fixed paraffin-embedded tissue (FFPET).
The cobas PIK3CA Test is based on two major processes: (1) manual sample preparation to obtain genomic DNA from FFPET; and (2) PCR amplification and detection of target DNA using complementary primer pairs and oligonucleotide probes labeled with fluorescent dyes. The test is designed to detect R88Q in exon 1, N345K in exon 4, C420R in exon 7, E542K, E545X (E545A, E545D^*, E545G, and E545K), Q546X (Q546E, Q546K, Q546L, and Q546R) in exon 9, and M1043I^†, H1047X (H1047L, H1047R, and H1047Y), and G1049R in exon 20 when the percent mutation is 5% or greater. Mutation detection is achieved through PCR analysis with the cobas z 480 analyzer. A mutant control and a negative control are included in each run to confirm the validity of the run.

Sample Preparation

FFPET samples are processed and genomic DNA isolated using the cobas^® DNA Sample Preparation Kit, a generic manual sample preparation based on nucleic acid binding to glass fibers. A deparaffinized 5 μm section of an FFPET sample is lysed by incubation at an elevated temperature with a protease and chaotropic lysis/binding buffer that releases nucleic acids and protects the released genomic DNA from DNases. Subsequently, isopropanol is added to the lysis mixture that is then centrifuged through a column with a glass fiber filter insert. During centrifugation, the genomic DNA is bound to the surface of the glass fiber filter. Unbound substances, such as salts, proteins and other cellular impurities, are removed by centrifugation. The adsorbed nucleic acids are washed and then eluted with an aqueous solution. The amount of genomic DNA is spectrophotometrically determined and adjusted to a fixed concentration to be added to the amplification/detection mixture. The target DNA is then amplified and detected on the cobas z 480 analyzer using the amplification and detection reagents provided in the cobas PIK3CA Test kit.

PCR Amplification

Target Selection

The cobas PIK3CA Test kit uses a pool of primers that define specific base-pair sequences that range from 85 to 155 base pairs long in PIK3CA exons 1, 4, 7, 9, and 20. An additional primer pair targets a conserved 167 base pair region in exon 3 of the PIK3CA gene to provide a full process control for sample adequacy, extraction and amplification. Amplification occurs only in the regions of the PIK3CA gene between the primers; the entire PIK3CA gene is not amplified.

Target Amplification

A derivative of Thermus species Z05-AS1 DNA polymerase is utilized for target amplification. First, the PCR reaction mixture is heated to denature the genomic DNA and expose the primer target sequences. As the mixture cools, the upstream and downstream primers anneal to the target DNA sequences. The Z05-AS1 DNA polymerase, in the presence of divalent metal ion and excess dNTP, extends each annealed primer, thus synthesizing a second DNA strand. This completes the first cycle of PCR, yielding a double-stranded DNA copy which includes the targeted base-pair regions of the PIK3CA gene. This process is repeated for a number of cycles, with each cycle effectively doubling the amount of amplicon DNA.

Automated Real-time Mutation Detection

The cobas PIK3CA Test utilizes real-time PCR technology. Each target-specific, oligonucleotide probe in the reaction is labeled with a fluorescent dye that serves as a reporter, and with a quencher molecule that absorbs (quenches) fluorescent emissions from the reporter dye within an intact probe. During each cycle of amplification, probe complementary to the single-stranded DNA sequence in the amplicon binds and is subsequently cleaved by the 5' to 3' nuclease activity of the Z05-AS1 DNA Polymerase. Once the reporter dye is separated from the quencher by this nuclease activity, fluorescence of a characteristic wavelength can be measured when the reporter dye is excited by the appropriate spectrum of light. Four different reporter dyes are used to detect the mutations targeted by the test. Amplification of the targeted PIK3CA sequences are detected independently across three reactions by measuring fluorescence at the four characteristic wavelengths in dedicated optical channels.

Selective Amplification

Selective amplification of target nucleic acid from the sample is achieved in the cobas PIK3CA Test by the use of AmpErase (uracil-Nglycosylase)
enzyme and deoxyuridine triphosphate (dUTP)¹. The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine but not DNA containing thymidine. Deoxyuridine is not present in naturally occurring DNA but is always present in amplicon due to the use of dUTP in addition to deoxythymidine triphosphate as one of the nucleotide triphosphates in the Master Mix reagents; therefore, only amplicon contains deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by AmpErase enzyme prior to amplification of the target DNA. The AmpErase enzyme, which is included in the Master Mix reagents, catalyzes the cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the C1-position. When heated in the first thermal cycling step at alkaline pH, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme is inactive at temperatures above 55ºC, i.e., throughout the thermal cycling steps, and therefore does not destroy target amplicon.

^* For the E545D amino acid change, only the nucleotide change c.1635G>T mutation is detected by the test.
^† For the M1043I amino acid change, only the nucleotide change c.3129G>T mutation is detected by the test.", "Language": "en", "Country": "XG", "Code": "Principle" }, { "Name": "Content", "Value": "

cobas® PIK3CA Mutation Test  (M/N: 06523013190)	24 Tests
	PIK3CA MMX-1 (PIK3CA Master Mix 1; Cap with White Button)	2 x 0.48 mL
	PIK3CA MMX-2 (PIK3CA Master Mix 2; Cap with Gold Button)	2 x 0.48 mL
	PIK3CA MMX-3 (PIK3CA Master Mix 3; Cap with Teal Button)	2 x 0.48 mL
	MGAC (Magnesium acetate; Cap with Yellow Button)	6 x 0.2 mL
	PIK3CA MC (PIK3CA Mutant Control; Cap with Red Button)	6 x 0.1 mL
	DNA SD (DNA Specimen diluent)	2 x 3.5 mL

", "Language": "en", "Country": "XG", "Code": "Content" } ] } } ] }