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Biotech-Catalog Molecular Biology Testing |
Molecular Biology Testing | | Molecular biology assays are an integral part of a majority of current biosafety testing programs. AppTec offers state-of-the-art nucleic acid-based molecular detection technologies for the evaluation of the purity of biological materials, for the characterization of molecular constructs, and for the detection and identification of adventitious contaminating agents. AppTec’s molecular technology is applicable to any study where the detection, characterization or quantification of a nucleic acid in biological material is required. This broad capability spans such areas as cell banks, viral stocks, gene therapy products (viral and non-viral), DNA vaccines, recombinant proteins, monoclonal antibodies, transgenic plants and animals, expression analysis, and drug efficacy studies with antiviral or anticancer agents. Molecular biology testing services include an array of PCR-based assays for detection and quantification of specific nucleic acid sequences, detection and quantification of residual contaminating DNA with hybridization or qPCR, and reverse transcriptase activity detection. Standard molecular protocols also include plasmid preparation and characterization, Northern and Southern blotting, restriction enzyme analysis, and DNA cloning and sequencing. These techniques are also available as part of custom projects. The molecular biology department provides the detection technology for AppTec’s biodistribution program. All animal work is performed at AppTec’s St. Paul facility and tissues for analysis are carefully catalogued and transferred to the molecular biology department at the Camden facility for nucleic acid extraction and quantitation by qPCR. Studies are performed under GLP or GMP conditions (unless R&D is requested), and all data are reviewed and verified by AppTec’s Quality Assurance department. Members of the QA department also perform critical step inspections for the GLP assays. AppTec specializes in the development and validation of custom assays and the knowledgeable staff maintains an ongoing, interactive collaboration with the client. AppTec’s staff is available to consult in the design of specific protocols and in the development of specific assays tailored to particular applications.
In this section
| | Polymerase Chain Reaction (PCR) Tests Back to Top | | Polymerase Chain Reaction (PCR) is a selective and sensitive method for the direct detection of nucleic acid sequences. The procedure and its acceptance as a method for detecting extremely low levels of genetic material are well documented. Enzymatic amplification of the specific DNA sequences is accomplished using selected oligonucleotide primers unique to the target. AppTec has developed two types of detection schemes using PCR. Using the Roche LightCycler and fluorescent oligo probes, quantitative real time PCR (qPCR) can be performed to generate a rapid quantitation of the number of copies or mass of a target nucleic acid sequence. Alternately, in our standard GLP/GMP limit tests, Southern blotting is also used with oligonucleotide probes specific to the internal sequences of the amplified regions to detect PCR products. Inhibition of PCR reactions due to contaminating material can be assessed by adding positive control DNA to prepared test articles before performing the PCR assay. Dedicated laboratory facilities and exacting procedures help ensure the high reliability of the testing results. Contamination of test articles with amplicons, which can lead to false positive results, is minimized by physical separation of each process. A dedicated clean room (free of specimens or amplified products) is used to prepare the amplification reagents. For each amplification reagent mix prepared there is a reagent-only control that is amplified. Additionally, in most of the protocols, dTTP is replaced with dUTP and the enzyme Uracil-N-Glycosylase is added to the reaction. This enzyme helps reduce any possible carryover contamination. The processing of the test articles is performed in a separate area and negative controls are processed concurrently with the test article. The negative controls act as sentinels. The amplification and detection area is also separated from reagent preparation or specimen preparation rooms. All assays can be performed at three levels – Research, GLP or cGMP – and each follows strictly the appropriate requirements for regulatory compliance. TURNAROUND TIME
Research tests have a 2-3 week turnaround time and research final reports are not reviewed by AppTec’s QA department. GLP and cGMP tests have a turnaround time fo 3-4 weeks depending on the assay. SAMPLE SUBMISSION REQUIREMENTS
Sample submission requirements vary depending on the nature of the test article. For cells, the minimum requirement is 1 X 106 cells. For fluids and solids, consultation with the Study Director or Client Services is recommended to ensure that appropriate amounts of material are submitted. SHIPPING CONDITIONS
The recommended shipping conditions are overnight, frozen on dry ice. However, growing cells in growth medium at ambient temperature or cryopreserved cells and/or other sample formats are acceptable. Again, consultation with Client Services is recommended to ensure appropriate shipping and preservation. | | Adventitious Agent Detection by PCR Back to Top | | Mycoplasma PCR Program Back to Top | | AppTec has developed a series of PCR assays designed to detect mycoplasma DNA in a test sample. These assays range in complexity from simple, rapid assays using ethidium stained gels to more complex assays that include Southern blotting and probing with internal oligonucleotide probes. All mycoplasma PCRs use a nested approach with two PCR amplifications employing two primer combinations, and employ primers and probes directed at a conserved ribosomal RNA gene sequence. All mycoplasma PCRs so far have been set up as limit tests, however development of quantitative real time PCRs for mycoplasma is ongoing. These primers and probes have been shown by experimentation to detect the most critical mycoplasma DNAs and by database homology searches align with over 60 known mycoplasma/acholeplasma species. NOTE: Sample container/volume and shipment temperature are at client’s discretion. | 39083 | Mycoplasma DNA is amplified and analyzed by slot blot with probing using an internal oligonucleotide. Assay sensitivity = 1000 copies. |
Detection of Mycoplasma DNA by PCR (Research)
Turnaround Time:
2-3 weeks
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| 30645 | Mycoplasma DNA is amplified and analyzed by ethidium bromide gel electrophoresis. Assay sensitivity = 1000 copies. N.B.: Samples received by Wednesday morning will have preliminary results by Friday afternoon. |
Detection of Mycoplasma DNA by PCR (GLP-Rapid)
Turnaround Time:
See N.B.
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| 30326 | Mycoplasma DNA is amplified and analyzed by Southern blotting with probing using an internal oligonucleotide. Assay sensitivity = 100 copies. |
Detection of Mycoplasma DNA by PCR (GLP)
Turnaround Time:
3 weeks
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| 33326 | Mycoplasma DNA is amplified and analyzed by Southern blotting with probing using an internal oligonucleotide. Assay sensitivity = 100 copies. |
Detection of Mycoplasma DNA by PCR (cGMP)
Turnaround Time:
3 weeks
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| | f | | Residual DNA Testing Program Back to Top | | AppTec’s Molecular Biology department has extensive experience testing biological materials for the presence of residual DNA. Offering several state-of-the-art quantitative PCR and hybridization assays, AppTec provides the highest level of residual DNA testing services. The hybridization assays offered are fully validated to fulfill ICH guidelines as limit tests. The qPCR assays are fully validated to fulfill ICH guidelines as quantitative tests (including accuracy, precision, limit of quantitation, range, linearity, specificity and robustness). AppTec added a Residual DNA Sizing protocol in response to an FDA letter to vaccine manufacturers that requires sizing information for residual DNA in vaccine products. REGULATORY COMPLIANCE
- Testing services range from Research to cGMP for Finished Pharmaceutical (21 CFR Part 211).
- Raw materials are obtained from approved vendors whose products meet required specifications.
- Certificates of Analysis are maintained for specific lots of reagents used in testing. SPECIES DETECTED
AppTec’s technology is able to detect residual DNA from multiple species, including human (several cell lines), mouse, hamster, chicken, dog, yeast, pig, monkey, cow and E. coli. |
Residual DNA - Slot Blot Hybridization
AppTec’s slot blot hybridization assays meet or exceed current U.S. and foreign regulatory requirements to detect residual cellular or plasmid DNA in biological products. In the slot blot assay, samples are treated with proteinase K and detergent and then processed to extract DNA. Multiple process and DNA spike/recovery controls are included to internally monitor the performance of each assay. DNA is bound to a nylon membrane and detected by hybridization with a homologous, species-specific 32P labeled probe. The blot is washed to remove unbound probe and exposed to x-ray film to obtain an autoradiogram. 30029 GLP
33029 cGMP | Residual DNA is detected by molecular hybridization using a radiolabeled homologous DNA probe. Two aliquots of the test article are processed by proteinase K digestion, multiple phenol extractions and ethanol precipitation. Process controls are run in parallel with the test article. The DNA concentration in the unspiked test article is evaluated visually by comparing the hybridization signal of the unspiked test article with those of the positive control DNA standards. Assay sensitivity is typically ≤10 pg DNA. Notes
· Specify amount of protein to be extracted (usually equal to one dose), protein concentration and species of DNA probe to be used.
· Volumes up to 5 ml/sample can be extracted. |
Detection of Contaminating DNA in Biological Samples
Turnaround Time:
4 weeks Sample Size:
Equivalent of 4 doses Shipment Temperature:
As appropriate |
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30062 GLP
33062 cGMP | Residual DNA is detected by molecular hybridization using a radiolabeled homologous DNA probe. Two aliquots of the test article are processed by proteinase K digestion, multiple phenol extractions and ethanol precipitation. Process controls are run in parallel with the test article. The DNA concentration in the unspiked test article is evaluated visually by comparing the hybridization signal of the unspiked test article with those of the positive control DNA standards. Assay sensitivity is typically ≤10 pg DNA. Notes
· Specify amount of protein to be extracted (usually equal to one dose), protein concentration and species of DNA probe to be used.
· Volumes up to 5 ml/sample can be extracted. |
Detection of Contaminating DNA with Two Spiked Samples
Turnaround Time:
4 weeks Sample Size:
Equivalent of 4 doses Shipment Temperature:
As appropriate |
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30095 GLP
33095 cGMP | Residual DNA is detected by molecular hybridization using radiolabeled homologous DNA probes. Six replicates of the test article are processed by proteinase K digestion, multiple phenol extractions and ethanol precipitation. Process controls are run in parallel with the test article. Two replicates are unspiked, two are spiked with 10 pg DNA, and two are spiked with 50 pg DNA to provide data on DNA recovery through the multi-step extraction and precipitation procedure. The DNA concentration in the unspiked test article is evaluated visually by comparing the hybridization signal of the unspiked test article with those of the positive control DNA standards. Assay sensitivity is typically ≤10 pg DNA. Notes
· Specify amount of protein to be extracted (usually equal to one dose), protein concentration and species of DNA probe to be used.
· Volumes up to 5 ml/sample can be extracted. |
Detection of Contaminating DNA with Two Spiked Samples in Duplicate
Turnaround Time:
4 weeks Sample Size:
Equivalent of 6 doses Shipment Temperature:
As appropriate |
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| 30639 cGMP | Residual DNA is detected by molecular hybridization using a radiolabeled homologous DNA probe. Two aliquots of the test article are processed by proteinase K digestion, multiple phenol extractions and ethanol precipitation. Process controls are run in parallel with the test article. The DNA concentration in the unspiked test article is evaluated visually by comparing the hybridization signal of the unspiked test article with those of the positive control DNA standards. Assay sensitivity is typically ≤10 pg DNA. Notes
· Specify amount of protein to be extracted (usually equal to one dose), protein concentration and species of DNA probe to be used.
· Volumes up to 5 ml/sample can be extracted. |
Detection of Contaminating DNA with Two Spiked Samples
(Client Choice)
Turnaround Time:
4 weeks Sample Size:
Equivalent of 4 doses Shipment Temperature:
As appropriate |
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Residual DNA - qPCR
qPCR for residual DNA offers significant improvements in specificity and sensitivity. qPCRs are set up as quantitative tests and allow limits of quantitation of sub picogram amounts of residual DNA. The assays also exhibit higher specificity due to the species-specific homologous primer/probe combinations. DNA is prepared for qPCR in an identical manner to that used for the slot blot assay and includes multiple process and spike recovery controls. GLP cGMP | Quantitation of Residual CHO DNA by qPCR | 30687 | 30688 | | Quantitation of Residual Human DNA by qPCR | 30685 | 30686 | | Quantitation of Residual Mouse DNA by qPCR | 30689 | 30690 | | Quantitation of Residual Porcine DNA by qPCR | 30729 | To Be Developed |
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Residual DNA - DNA Sizing
In response to a letter from the FDA office of Vaccines (Letter to Sponsors Using Vero cells as a Cell Substrate for Investigational Vaccines. CBER Division of Vaccines. March 12, 2001), AppTec developed and qualified an assay for the measurement of the size of contaminating DNA in a sample. Residual DNA size in a sample is measured by Southern blotting and probing the blot with a species-specific total DNA probe. Controls for extraction and DNA transfer efficiency of fragments of different sizes are essential components of a sizing protocol and are included in this assay. 30676 GLP
30668 cGMP | This assay is to be run in parallel with the assays for the determination of Residual DNA mass. DNA of known mass and size is spiked into replicates of the test sample to give a determination of the efficiency and size profile of recovered DNA. DNA samples of known mass and size are also included in addition to those spiked into the test sample. Total Residual DNA is extracted from the samples, separated by agarose gel electrophoresis, and Southern blotted. For a determination of recovery efficiency of different size fragments and recovery size profile, the DNA of known mass and size is probed with a specific homologous probe. For a determination of contaminating DNA size, the test samples are probed with a probe consisting of total DNA from the species of interest. Scanning densitometry is used to generate values for DNA size and DNA recovery and transfer efficiency of different DNA size fragments. |
Sizing of Contaminating DNA by Southern Blotting
Turnaround Time:
6 weeks Container/Volume:
At client’s discretion Shipment Temperature:
As appropriate |
| | f | | Reverse Transcriptase Assays Back to Top |
| 30008 | This assay detects the presence of human and animal retroviruses in cultured cells. The assay quantitates the incorporation of radiolabeled nucleotides into cDNA which is copied from a synthetic template. Each test article is concentrated by ultracentrifugation and incubated with individual reaction mixtures containing a synthetic oligo (dT)–poly (rA) template and either Mn2+ or Mg2+ ions to discriminate between retroviruses having different cation preference. Notes
· Provide cell-free supernatant from culture in exponential growth, ascites fluid or production lot material.
· Sample should be shipped in sealed, leakproof plastic vials. |
Reverse Transcriptase Assay
Turnaround Time:
3 weeks Container/Volume:
2 x 3 ml Shipment Temperature:
Frozen on dry ice |
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| 30094 | This assay detects the presence of human and animal retroviruses in cultured cells. The assay quantitates the incorporation of radiolabeled nucleotides into cDNA which is copied from a viral template. Each test article is incubated in individual reaction buffers containing either Mn2+ or Mg2+ ions to discriminate between retroviruses having different cation preferences. In addition, the test article is incubated in the presence of the virus-positive controls to assess any effect of the test article on the positive control response. Notes
· Provide cell-free supernatant from culture in exponential growth, ascites fluid or production lot material.
· Sample should be shipped in sealed, leakproof plastic vials. |
Reverse Transcriptase Assay, Including Positive Control
Dilution Series
Turnaround Time:
3 weeks Container/Volume:
4 x 3 ml Shipment Temperature:
Frozen on dry ice |
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| 30039 | This assay detects the presence of human and animal retroviruses in cultured cells. The assay quantitates the incorporation of radiolabeled nucleotides into cDNA which is copied from a synthetic template. Each test article is concentrated by ultracentrifugation and incubated with individual reaction mixtures containing a synthetic oligo (dT)-poly(rA) template or a synthetic oligo (dT)-poly(rA) template and either Mn2+ or Mg2+ ions to discriminate polymerases between endogenous cellular DNA-dependent DNA polymerase and viral RNA-dependent DNA polymerase, and retroviruses having different cation preference. Notes
· Provide cell-free supernatant from culture in exponential growth, ascites fluid or production lot material.
· Sample should be shipped in sealed, leakproof plastic vials. |
Reverse Transcriptase Assay Utilizing Two Templates
Turnaround Time:
3 weeks Container/Volume:
4 x 3 ml Shipment Temperature:
Frozen on dry ice |
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30611 GLP
33611 cGMP | The PBRT (PERT) assay is a sensitive method for detecting the presence of enzymes with reverse transcriptase activity. The method can detect enzyme activity from as few as 10 molecules of reverse transcriptase, which is about one million fold more sensitive than standard enzymatic reverse transcriptase assays. The assay detects enzymes with either magnesium or manganese divalent cation requirements and is capable of discriminating activity due to contaminating cellular DNA polymerases from authentic reverse transcriptase molecules. The Center for Biologics Evaluation and Research (CBER) issued a letter, on December 14, 1998, to inform manufacturers of certain viral vaccine products that this test will be requested. Notes
· Provide cell-free supernatant from culture in exponential growth, ascites fluid or production lot material.
· Sample should be shipped in sealed, leakproof plastic vials. |
PCR-Based Reverse Transcriptase (PBRT) Assay
Turnaround Time:
4 weeks Container/Volume:
1 x 1 ml Shipment Temperature:
Frozen on dry ice |
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