Development and Characterization of Synthetic Standards: HCV RNA Susan E. Bromley, Ph.D. Director, Product Development Bayer Healthcare Diagnostics 24.

Slides:



Advertisements
Similar presentations
PCR, Gel Electrophoresis, and Southern Blotting
Advertisements

Jean-Michel PAWLOTSKY
Paris, May 2004SoGAT XVII SoGAT and the Development of Standards Harvey Holmes Division of Retrovirology NIBSC, UK.
Linearity Panels HIV RNA, HCV RNA, HBV DNA, and CMV DNA
XVIII SoGAT Washington 24 May 2005 SoGAT and HIV NAT Standards - 2 nd International Standard for HIV-1 RNA Harvey Holmes*, Clare Davis* and Alan Heath**
Structures of the Purine and Pyrimidine Bases Example of the Structure of a Nucleotide Base Nucleoside Nucleotide.
Tools for Molecular Biology Amplification. The PCR reaction is a way to quickly drive the exponential amplification of a small piece of DNA. PCR is a.
Differential Gene Expression in the Gastrula of Xenopus Laevis Differential Gastrula mRna – DG mRNA Transcribed during Gastrula stage; Selectively used.
Additional Powerful Molecular Techniques Synthesis of cDNA (complimentary DNA) Polymerase Chain Reaction (PCR) Microarray analysis Link to Gene Therapy.
Synthetic Biology Crash Course DAY ONE. Pre-Assessment 1:00-1:10.
Quantifying Sample DNA. Definition Quantifying DNA: a technique to calculate the quantity (weight) of DNA (deoxyribonucleic acid) in a sample. Using a.
Kyiv, TRAINING WORKSHOP ON PHARMACEUTICAL QUALITY, GOOD MANUFACTURING PRACTICE & BIOEQUIVALENCE Validation of Analytical Methods Used For Bioequivalence.
Molecular Cell Biology Fifth Edition Chapter 9: Molecular Genetic Techniques and Genomics Copyright © 2004 by W. H. Freeman & Company Harvey Lodish Arnold.
Variants of PCR Lecture 4
Einstein-Montefiore CFAR Virology Core Director: Dr. Ganjam V. Kalpana, Ph.D.
Analysis of Transgenic Plants. 1.Regeneration on Selective Medium Selectable Marker Gene.
Bayer Molecular Customer HIV-1 Quantitation QA Scheme.
TOPICS IN (NANO) BIOTECHNOLOGY Lecture 7 5th May, 2006 PhD Course.
HIV, HCV, and HBV NAT Controls Formulation, Stability and Performance Mark Manak BBI Diagnostics, Inc. A Division of SeraCare Life Sciences, Inc. SoGAT.
Biotechnology. DNA technology DNA diagnostics DNA therapy.
NIST Standard Reference Material Project: Pure DNA Standard for Cytomegalovirus SoGAT XX Warsaw, Poland June 13,2007 Marcia Holden.
Poor Reproducibility of HIV­1 Low-level Viraemia Results with 3 Commercial Real-time PCR Assays Jean Ruelle 1, Laurent Debaisieux 2, Ellen Vancutsem 3,
Food and Drug Administration Center for Biologics Evaluation and Research The Office of Cellular, Tissue, and Gene Therapies Web Seminar Series presents:
DNA. A. Terminology A. Terminology Chromosomes- strands of genetic material Chromosomes- strands of genetic material Genes- Fundamental unit of heredity.
Chapter 20 DNA Technology and Genomics
Chapter 19 – Molecular Genetic Analysis and Biotechnology
 It is the methods scientist use to study and manipulate DNA.  It made it possible for researchers to genetically alter organisms to give them more.
-The methods section of the course covers chapters 21 and 22, not chapters 20 and 21 -Paper discussion on Tuesday - assignment due at the start of class.
CE MARKING OF IVDDs - the NIBSC perspective Morag Ferguson Division of Virology.
Biotechnology Packet #12 Chapter #9. Introduction Since the 1970’s, humans have been attempted to manipulate and modify genes in a way that was somewhat.
Genetic modification techniques. Tools of biotechnology Collect DNA Restriction enzymes –Blunt end –Sticky end Ligase enzymes Cloning DNA carriers –Bacteria.
Library screening Heterologous and homologous gene probes Differential screening Expression library screening.
 It is the methods scientist use to study and manipulate DNA.  It made it possible for researchers to genetically alter organisms to give them more.
Yi-Chen Yang, Yu-Hsuan Chen, Show-Lan Chiu, Hwei-Fang Cheng Drug Biology Division, Bureau of Food and Drug Analysis Department of Health, Taiwan, ROC National.
Tools of Human Molecular Genetics. ANALYSIS OF INDIVIDUAL DNA AND RNA SEQUENCES Two fundamental obstacles to carrying out their investigations of the.
Manipulation of DNA. Restriction enzymes are used to cut DNA into smaller fragments. Different restriction enzymes recognize and cut different DNA sequences.
1 Chapter 2: DNA replication and applications DNA replication in the cell Polymerase chain reaction (PCR) Sequence analysis of DNA.
Monitoring the Performance of Nucleic Acid Tests using Data Generated from EDCNet and DigitalPT Wayne Dimech, Darren Jardine, Thu-Anh Pham and the staff.
Chapter 6 PCR and in vitro Mutagenesis A. Basic features of PCR 1. PCR is a cell-free method of DNA cloning standard PCR reaction is a selective DNA amplification.
Armored RNA Quant ™ Development of Armored RNA ® as a Primary Standard June 5, 2003 Cindy WalkerPeach, Ph.D. Director of Research and cGMP Operations Ambion.
 DNA (gene mutations, paternity, organs compatibility for transplantations)  RNA  Proteins (gene expression)
History of ILC Involvement in NAT Standardization 1998: Inter-Organization Discussion for the Establishment of Standard Reference Material for Nucleic.
Performance of Chiron Quantitative and Qualitative HBV PCR Assay and Confirmation of HBV Yield Cases Yiu-Lian Fong, Ph.D, Associate Director June 13, 2007.
Chapter 20 DNA Technology and Genomics. Viruses have restriction enzymes to attack and destroy invading viral DNA. Restriction enzymes cut DNA at specific.
Molecular Testing and Clinical Diagnosis
International Scientific Workshop on the Standardization of Genome Amplification Techniques for the Safety Testing of Blood, Tissues and Organs With Regard.
Molecular Tools. Recombinant DNA Restriction enzymes Vectors Ligase and other enzymes.
Recombinant DNA Technology. DNA replication refers to the scientific process in which a specific sequence of DNA is replicated in vitro, to produce multiple.
DNA Technology & Genomics
KINETICS OF PROMOTER ESCAPE VARIES AS A FUNCTION OF KCl CONCENTRATION Sophiya Karki, Elina Shrestha and Lilian M. Hsu* Program in Biochemistry, Mount Holyoke.
Confidential Extending the Armored RNA Technology to DNA Cindy WalkerPeach, Ph.D. Director, Development and Operations Ambion Diagnostics BSI EN ISO9001:2000.
RECOMBINANT DNA DNA THAT CONTAINS DNA SEGMENTS OR GENES FROM DIFFERENT SOURCES. DNA TRANSFERRED FROM ONE PART OF A DNA MOLECULE TO ANOTHER, FROM ONE CHROMOSOME.
Progress on Study to Validate Synthetic Materials for Use as Calibrators and References SoGAT XIX Bern, 14 June 2006 Roberta M. Madej Roche Molecular Diagnostics.
Difficulties with DNA 1. 1.One cell normally provides too little material for study Gene cloning Polymerase Chain Reaction (PCR) 2. 2.There are often.
DNA Isolation. Nucleic Acid Structure & Function DNA & RNA are composed of Nucleotides A nucleotide consists of three covalently-linked parts: –A nitrogen.
Rajan sharma.  Polymerase chain reaction Is a in vitro method of enzymatic synthesis of specific DNA sequences.  This method was first time developed.
C H-NS (nM) 14 nM FIS28 nM FIS56 nM FISno FIS Figure S1. FIS and H-NS can simultaneously interact with cspA promoter.
PCR and Gel Electrophoresis
PLANT BIOTECHNOLOGY & GENETIC ENGINEERING (3 CREDIT HOURS)
DNA Technology Packet #27.
Relationship between Genotype and Phenotype
Roche Molecular Diagnostics
Volume 3, Issue 1, Pages (January 1999)
DNA Technology Packet #50 Chapter #20.
Transcriptional Fidelity and Proofreading by RNA Polymerase II
Volume 10, Issue 5, Pages (November 2002)
Frpo: A Novel Single-Stranded DNA Promoter for Transcription and for Primer RNA Synthesis of DNA Replication  Hisao Masai, Ken-ichi Arai  Cell  Volume.
Can plasmids replicate in different bacteria?
Relationship between Genotype and Phenotype
Volume 3, Issue 1, Pages (January 1999)
Presentation transcript:

Development and Characterization of Synthetic Standards: HCV RNA Susan E. Bromley, Ph.D. Director, Product Development Bayer Healthcare Diagnostics 24 May 2005

Goals for Synthetic Reference Standards Provide reference standard that: – –has a confirmed identity – –can be reproducibly manufactured – –can be quantitated by an independent, traceable method – –can be independently monitored for stability – –provides a quantitation anchor for the VERSANT® HCV bDNA Assay standardization scheme. Validate use of reference standard: – –reproducible quantitations between reference standard lots and kit lots

Purify Cloned DNAConfirm identity by sequencing In vitro transcription reaction Purify Transcript UV Scan Denaturing gel Digest Transcript Sample Hyperchromicity Phosphate Determination Aliquot and store Tracer incorporation Stability monitored Manufacturing StepsQualification Steps

Manufacturing and Qualification Cloned plasmid DNA is used as a template for in vitro transcription to produce RNA transcript – –identity of clone confirmed by sequencing In vitro transcription reaction using T7 RNA polymerase – –incorporation of very low-level radioactive nucleoside precursor – –denaturing gel analysis for full-length product

Independent RNA Quantitation Three analytical methods – –A 260nm – –Hyperchromatic Shift – –Phosphate Determination Three methods must agree within 20% Value from phosphate determination used – –Purified RNA digested to nucleosides and phosphates with SVPD and CIP – –Phosphate complexed with ammonium molybdate – –A 360nm calibrated to NIST phosphorus anion standard reference material

Stability: Northern Blot Analysis of Reference HCV RNA RNA Marker (Kb) Full Length Transcripts RNA Markers (Kb) Degraded RNA

Reference Standard transcript of HCV 5’NC and Core gene Quantification of Kit Calibrators Quantification of QC Panels Quantification of WHO Standard Use of HCV Reference Standard at Bayer Diagnostics Quantification Kit Controls Quantification Patient Samples

Validations Process validationProcess validation –Three diverse lots manufactured and qualified according to procedures –All specifications exceeded Standardization validationStandardization validation –Three lots of reference standard and three kit lots –Value assignment of kit calibrators –4 replicates of each calibrator in 6 assays, 3 operators and instruments –Variation in value assignment of the kit calibrators due to all factors was less than 5%.

Relationship to WHO Standard Perform value assignment experiment as used to assign quantitations to QC panels and kit calibrators Each value assignment experiment for the WHO Standard includes 9 assays with 4 replicates of each of 6 dilutions WHO value assignment was expanded to include 7 different VERSANT® HCV RNA 3.0 (bDNA) kit lots and 4 lots of HCV Reference Standard 5.2 bDNA copies/WHO IU, 8.3%CV

Conclusions Synthetic HCV Reference Standard RNA provides stable quantitation anchor for the VERSANT® HCV RNA 3.0 (bDNA) Assay.Synthetic HCV Reference Standard RNA provides stable quantitation anchor for the VERSANT® HCV RNA 3.0 (bDNA) Assay. In use since 2000In use since 2000 Same model for standardization used for HIV and HBV assays.Same model for standardization used for HIV and HBV assays.