Systematic Analysis of single cells by PCR
|
|
- Ralf Summers
- 6 years ago
- Views:
Transcription
1 Systematic Analysis of single cells by PCR Wolfgang Mann 27th March 2007, Weihenstephan
2 Overview AmpliGrid Technology Examples DNA Forensics Single Cell Sequencing Polar Body Diagnostics Single Cell RT PCR Advalytix AG 2007 Page 2
3 Challenges in qpcr Technical sample prep robust sample analysis when dealing with small volumes optimization of fluorescent dyes, development of alternative systems biochemistry of enzymes involved statistical procedures (normalization, optimization, standardization) workflow aspects, post amplification analysis, intrumentation Functional robust assay development R&D applications (sirna, etc) drug discovery Advalytix AG 2007 Page 3
4 towards genetic single cell analysis Analysis of small numbers of cells pools vs individuals Advalytix AG 2007 Page 4
5 towards genetic single cell analysis Analysis of small numbers of cells pools vs individuals Advalytix AG 2007 Page 5
6 towards genetic single cell analysis Examples for single or little cell numbers mouse morulae: RT-PCR analysis of single blastomeres (May et al. 2007) human polar bodies: analysis of chromosomal disorders (Hagen-Mann et al. 2005) Advalytix AG 2007 Page 6
7 towards genetic single cell analysis Examples for single or little cell numbers single blood cells: validation of forensic methods (Proff et al. 2006) single lymphocytes: mtdna sequence analysis (Lutz-Bonengel et al. 2006, 2007) Advalytix AG 2007 Page 7
8 AmpliGrid a new platform for sc amplification AmpliGrid Advalytix AG 2007 Page 8
9 AmpliGrid optical control of the target cell single cells on the AmpliGrid (Hoechst staining): the goal is to amplify exactly two copies of a sequence nc nc Advalytix AG 2007 Page 9
10 AmpliGrid robotics Main important steps: pipetting of aqueous phase (1µl) overlay with covering solution (5µl) Advalytix AG 2007 Page 10
11 AmpliSpeed Cycler AmpliSpeed SlideCycler non-peltier based robot compatible modular setup Advalytix AG 2007 Page 11
12 AmpliGrid technology Summary AmpliGrid Technology: Volume: 1µl PCR solution low cost, low thermal mass Glass slide instead of MTP improved heat conductivity Automated pipetting high throughput applications Flat surface, glass slide optical control of the target AmpliSpeed HT cycling Advalytix AG 2007 Page 12
13 Example: redundant PCR Improved information content by redundant PCR applying a forensic STR kit to the AmpliGrid ½vol tube PCR, conventional CE analysis DNA sample (low amount) ½vol 1µl PCR 1µl PCR 1µl PCR 1µl PCR 1µl PCR CE analysis AmpliGrid Advalytix AG 2007 Page 13
14 Example: redundant PCR on forensic stains 25pg of DNA: comparison of conventional vs redundant LV PCR Tube 25µL 44 LV-PCRs Typing success could be improved in 40% of the DNA samples tested Proff et al., Int. Congress Series 1288: , 2006 Redundant PCR in 1µl could be a general approach to other applications as well Advalytix AG 2007 Page 14
15 Example: forensic PCR Improved sensitivity on the AmpliGrid platform Comparison of different amplification geometries, subsequent sequencing mtdna amplification using primers L29/H381 Lutz-Bonengel et al., Int. J. Legal Med 121: 68-73, 2007 Advalytix AG 2007 Page 15
16 Example: forensic PCR Improved sensitivity on the AmpliGrid platform in order to study heteroplasmy of mitochondrial DNA FACS on Ampli Grid PCR + sequencing in 1µl Lutz-Bonengel et al., in prep Advalytix AG 2007 Page 16
17 Example: single cell sequencing Simple workflow for single cell sequencing lymphocyte preparation by FACS (48 cells in < 30s) PCR amplification of mtdna adding ExoSAPit in order to get rid of PCR primers adding cycle sequencing kit (including seqeuncing primer) cycle sequencing transfer into MTP apply to CE and read out In a systematic study Lutz-Bonengel et al (2007) could show: 96 % of heteroplasmy occur within tissues 4 % occur within cells or within mitochondria Diagnostics: clonal expansion of mtdna haplotypes in leukemia patients can be analysed by mtdna analysis (Yao et al. 2007) Advalytix AG 2007 Page 17
18 Example: PCR on single sequences DNA Typing of single cells with forensic STR kits (ABI) different numbers of single cells as a model for distinct copy numbers systematic study on FACS sorted cells (lymphocytes) Proff et al. 2006; drawbacks: FACS sort not optimized no nucleus staining Basic data for single cell analysis from forensic stains Advalytix AG 2007 Page 18
19 Example: human polar body analysis The goal is to detect presence or absence of all human chromosomes in polar bodies after sample preparation by glass capillary. Advalytix AG 2007 Page 19
20 Example: human polar body analysis Workflow: Sample preparation under the microscope (optical control) Deposition of polar bodies onto the AmpliGrid Transfer to the molecular lab (optical control of the sample) PCR analysis Polar body analysis is an example for an application with: A limited number of cells is to be analysed Each of the cells has to be analysed There is no second shot AmpliGrid has turned out to be an excellent storage device Optical control is the advantage for quality control of the workflow Advalytix AG 2007 Page 20
21 Polar body analysis: the test The test routine is as follows: Deposition of the single cell onto the AmpliGrid Overall amplification of the genome in 1µl (reducing complexity) Distribution of this amplicon to a number of multiplex PCRs Detection of products in HT mode (PAA, LiCor, etc) 5 markers / chromosome designed test result for single cell amplification Advalytix AG 2007 Page 21
22 Polar body analysis: results PCR results on polar body analysis Advalytix AG 2007 Page 22
23 Expanding the number of markers / genome Polar body analysis: results Advalytix AG 2007 Page 23
24 principle of ABC Amplification Based Counting PCR success rate = # of successful sub-pcrs total # of sub-pcrs ABC hinges on the concept of redundant sub-pcrs which amplify sub-sequences located within a sequence whose copy number is to be counted The average success rate of a set of sub-pcrs and the copy number of a starting sequence (template) correspond very reliably: for a given PCR protocol, drop-outs occur once the copy number falls below a fixed threshold ( dropout regime ) ABC comprises a range of diagnostic tools to exploit this fundamental insight Advalytix AG 2007 Page 24
25 applications in human genetics Cause disease Copy number deviation causes medical condition Examples Turner syndrome (45,X0) Trisomies: 13, 18, 21 Deletions: Cri-du-Chat (5p), Wolf-Hirschhorn (4p) HER2 (breast cancer) P53 (tumor suppressor) Mark disease Copy number change in a noncoding section of DNA Presents pre-disposition to disease or indicates a medical condition Examples STRs as markers for LOH (loss of heterocygozity) -5p -17q Microsatellites (MIN): HNPCC (hereditary nonpolyposis colon cancer) Single cell analysis is needed in IVF, fetal cells in maternal blood, studying mosacism, CTC, etc... Advalytix AG 2007 Page 25
26 Example: RT-PCR RNA amplification on the AmpliGrid has been shown to be very sensitive. As an example, a 308bp fragment was amplified from a 3000b transcript using RT-PCR. As little as 10 copies can be detected. LS: MW standard 10 5 : RNA copies 10 4 : RNA copies 10 3 : 1000 RNA copies 10 2 : 100 RNA copies 10: 10 RNA copies 1: 1 RNA copy 0: negative cotrol Advalytix AG 2007 Page 26
27 Example: RT-PCR on CD4+ cells Cells have been FACS sorted after staining with an anti CD4-antibody Expression of CD4 mrna was shown in some of the single cells 310bp RT PCR product specific for CD4 mrna combination of immunophenotyping and genetic analysis of the identical cell: a very new field to exploit Advalytix AG 2007 Page 27
28 Example: complex RT PCR on mouse embryos Analysis of gene expression of single mouse embryos a set of genes has been chosen that are all expressed in the mouse embryo: DNA modification genes encoding DNA methyltransferases Dnmt3a, Dnmt3b, Dnmt1, Dnmt3l methyl-cpg binding domain proteins Mbd1, Mbd2, Mbd3 and Mbd4 DNA repair proteins Polb, Apex1, Lig1, Lig3 (active demethylation by base excision) control genes H3.3, Pou5f1 Advalytix AG 2007 Page 28
29 Example: complex RT PCR on mouse embryos Experimental set up: sensitivity test on total RNA analysis of morulae analysis of single blastocystes Multiplex RT PCR on 1000pg of total RNA, 14 genes, 12 replicates (left / right lane: molecular size marker) Advalytix AG 2007 Page 29
30 Example: complex RT PCR on mouse embryos Analysis of total mouse morulae A1, A2,... : different mouse morulae PBS, (EC), H2O: negative controls 50bp: molecular weight marker Advalytix AG 2007 Page 30
31 Example: complex RT PCR on mouse embryos Analysis of single mouse blastocystes M A1 A4 A6 A8 A9 A10 B1 B4 PCR RT PBS PBS (EC) H 2 0 A1, A2,..B1,. : different mouse blastocysts PBS, (EC), H2O, PCR, RT: negative controls M: molecular weight marker Preliminary work will be followed by systematic analysis and semiquantitative analysis of products (May et. al, 2007, in prep) Advalytix AG 2007 Page 31
32 summary AmpliGrid is a new platform for amplification reactions (PCR/RT-PCR) It has been designed for single cell use Optical features of the chip allow to qualify the target cells Different characteristics might be combined (immunophenotyping + PCR) AmpliGrid allows high throughput, systematic analysis of single cells Even other ideas are on the way: culture the cell on chip, stress the cell,.. Advalytix AG 2007 Page 32
33 contributions Advalytix thanks the following partners: GSF, Neuherberg Gerichtsmedizinisches Institut, Universität Köln Gerichtsmedizinischs Institut, Universität Freiburg Humangenetisches Institut, Universität Mainz Advalytix AG 2007 Page 33
Functional Genomics Research Stream. Research Meeting: June 19, 2012 SYBR Green qpcr, Research Update
Functional Genomics Research Stream Research Meeting: June 19, 2012 SYBR Green qpcr, Research Update Updates Alternate Lab Meeting Fridays 11:30-1:00 WEL 4.224 Welcome to attend either one Lab Log thanks
More informationNature Methods: doi: /nmeth Supplementary Figure 1
Supplementary Figure 1 Workflow for multimodal analysis using sc-gem on a programmable microfluidic device (Fluidigm). 1) Cells are captured and lysed, 2) RNA from lysed single cell is reverse-transcribed
More informationShaping Epigenetics Discovery
Shaping Epigenetics Discovery Kits, Assays, Controls, Inhibitors and Antibodies for DNA Methylation The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and
More informationFrom Liquid Biopsy and FFPE Samples to Results
From Liquid Biopsy and FFPE Samples to Results Fully automated purification, reliable quantification and bisulfite conversion of cell-free circulating DNA and DNA from FFPE samples Fully automated purification,
More informationFrom Liquid Biopsy and FFPE Samples to Results
www.autogen.com 774-233-3000 From Liquid Biopsy and FFPE Samples to Results Fully automated purification, reliable quantification and bisulfite conversion of cell-free circulating DNA and DNA from FFPE
More information6th Form Open Day 15th July 2015
6th Form Open Day 15 th July 2015 DNA is like a computer program but far, far more advanced than any software ever created. Bill Gates Welcome to the Open Day event at the Institute of Genetic Medicine
More informationChapter 20 Recombinant DNA Technology. Copyright 2009 Pearson Education, Inc.
Chapter 20 Recombinant DNA Technology Copyright 2009 Pearson Education, Inc. 20.1 Recombinant DNA Technology Began with Two Key Tools: Restriction Enzymes and DNA Cloning Vectors Recombinant DNA refers
More informationThe Polymerase Chain Reaction. Chapter 6: Background
The Polymerase Chain Reaction Chapter 6: Background PCR Amplify= Polymerase Chain Reaction (PCR) Invented in 1984 Applications Invention of PCR Kary Mullis Mile marker 46.58 in April of 1983 Pulled off
More informationINTRODUCTION TO REVERSE TRANSCRIPTION PCR (RT-PCR) ABCF 2016 BecA-ILRI Hub, Nairobi 21 st September 2016 Roger Pelle Principal Scientist
INTRODUCTION TO REVERSE TRANSCRIPTION PCR (RT-PCR) ABCF 2016 BecA-ILRI Hub, Nairobi 21 st September 2016 Roger Pelle Principal Scientist Objective of PCR To provide a solution to one of the most pressing
More informationNovel methods for RNA and DNA- Seq analysis using SMART Technology. Andrew Farmer, D. Phil. Vice President, R&D Clontech Laboratories, Inc.
Novel methods for RNA and DNA- Seq analysis using SMART Technology Andrew Farmer, D. Phil. Vice President, R&D Clontech Laboratories, Inc. Agenda Enabling Single Cell RNA-Seq using SMART Technology SMART
More informationLecture 9. Eukaryotic gene regulation: DNA METHYLATION
Lecture 9 Eukaryotic gene regulation: DNA METHYLATION Recap.. Eukaryotic RNA polymerases Core promoter elements General transcription factors Enhancers and upstream activation sequences Transcriptional
More informationQuantifiler Human DNA Quantification Kit Quantifiler Y Human Male DNA Quantification Kit
Product Bulletin Human Identification Quantifiler Human DNA Quantification Kit Quantifiler Y Human Male DNA Quantification Kit The Quantifiler kits produce reliable and reproducible results, helping to
More informationGene Expression on the Fluidigm BioMark HD
Gene Expression on the Fluidigm BioMark HD Overview Introduction to Fluidigm James Miller Advantages of the technology Running a Fluidigm gene expression project Paul Lacaze Assay design, chemistry, experimental
More informationSite directed mutagenesis, Insertional and Deletion Mutagenesis. Mitesh Shrestha
Site directed mutagenesis, Insertional and Deletion Mutagenesis Mitesh Shrestha Mutagenesis Mutagenesis (the creation or formation of a mutation) can be used as a powerful genetic tool. By inducing mutations
More informationExpressed genes profiling (Microarrays) Overview Of Gene Expression Control Profiling Of Expressed Genes
Expressed genes profiling (Microarrays) Overview Of Gene Expression Control Profiling Of Expressed Genes Genes can be regulated at many levels Usually, gene regulation, are referring to transcriptional
More informationFully Automated Library Quantification for Illumina Sequencing on the NGS STAR
Fully Automated Library Quantification for Illumina Sequencing on the NGS STAR Introduction Hamilton Robotics, an industry leader in liquid handling and laboratory automation equipment, has partnered with
More informationAIT - Austrian Institute of Technology
BIOMARKER DISCOVERY, BIOINFORMATICS, AND BIOSENSOR DEVELOPMENT Technology Experience AIT Austrian Institute of Technology Low-Emission Transport AIT - Austrian Institute of Technology Energy Health & Bioresources
More informationGreenMasterMix (2X) b i o s c i e n c e. G E N A X X O N b i o s c i e n c e. High ROX (500nM)
G:\products\productflyer\pcr\polymerasen\hotstart\manu_m3052_green_en.docx GreenMasterMix (2) High RO (500nM) qpcr master mix with fluorescence dye and passive reference dye Contact & Technical support
More informationMRC-Holland MLPA. Description version 15; 23 July 2015
SALSA MLPA probemix P125-B1 Mitochondria Lot B1-0312 B1-0609. Compared to the previous version of this product (lots A1-0606, A1-1105), four probes have been replaced and six extra mitochondrial probes
More informationAll your genetic analyses on a single instrument
GENOMELAB GEXP GENETIC ANALYSIS SYSTEM All your genetic analyses on a single instrument GENOMELAB GEXP GENETIC ANALYSIS SYSTEM All on a single instrument Perform your genetic assays on one instrument A
More informationDNA Workflow. Marine Biological Laboratory. Mark Bratz Applications Scientist, Promega Corporation. August 2016
DNA Workflow Marine Biological Laboratory Mark Bratz Applications Scientist, August 2016 Scientific Applications Support Mission Realize customer driven applications of Promega technologies to enhance
More informationMethods of Biomaterials Testing Lesson 3-5. Biochemical Methods - Molecular Biology -
Methods of Biomaterials Testing Lesson 3-5 Biochemical Methods - Molecular Biology - Chromosomes in the Cell Nucleus DNA in the Chromosome Deoxyribonucleic Acid (DNA) DNA has double-helix structure The
More informationHELINI Hepatitis B virus [HBV] Real-time PCR Kit (Genotype A to H)
HELINI Hepatitis B virus [HBV] Real-time PCR Kit (Genotype A to H) Quantitative In vitro diagnostics Instruction manual Cat. No: 8001-25/50/100 tests Compatible with: Agilent, Bio-Rad, Applied Bio systems
More informationSanger Sequencing Portfolio
Molecular Cloning Laboratories Sanger Sequencing Portfolio Significant Cost Savings vs ABI Uncompromised Performance Longer Read Length Longer Shelf Life No Recalibrations Necessary PAGE 1 BrightDye Terminator
More informationProtein and transcriptome quantitation using BD AbSeq Antibody-Oligonucleotide
Protein and transcriptome quantitation using BD AbSeq Antibody-Oligonucleotide technology and the 10X Genomics Chromium Single Cell Gene Expression Solution Jocelyn G. Olvera, Brigid S. Boland, John T.
More informationThe Polymerase Chain Reaction. Chapter 6: Background
The Polymerase Chain Reaction Chapter 6: Background Invention of PCR Kary Mullis Mile marker 46.58 in April of 1983 Pulled off the road and outlined a way to conduct DNA replication in a tube Worked for
More informationIntroducing: 3Zomy Aneuploidity Test
Introducing: 3Zomy Aneuploidity Test QF-PCR Life Technologies (India) Pvt. Ltd. 306, Aggarwal City Mall, Opposite M2K Pitampura, Delhi 110034 (INDIA). Ph: +91-11-42208000, 4220811, 42208222 Mobile: +91-9810521400
More informationComplete Sample to Analysis Solutions for DNA Methylation Discovery using Next Generation Sequencing
Complete Sample to Analysis Solutions for DNA Methylation Discovery using Next Generation Sequencing SureSelect Human/Mouse Methyl-Seq Kyeong Jeong PhD February 5, 2013 CAG EMEAI DGG/GSD/GFO Agilent Restricted
More informationRelative Fluorescent Quantitation on Capillary Electrophoresis Systems:
Application Note Relative Fluorescent Quantitation on the 3130 Series Systems Relative Fluorescent Quantitation on Capillary Electrophoresis Systems: Screening for Loss of Heterozygosity in Tumor Samples
More informationQuantiNova proven compatibility with various real-time cyclers in gene expression analysis
QuantiNova proven compatibility with various real-time cyclers in gene expression analysis QuantiNova SYBR Green and QuantiNova Probe PCR Kits were used on various real-time cyclers, and compared with
More informationAbsolute Human Telomere Length and Mitochondrial DNA Copy Number Dual Quantification qpcr Assay Kit (AHDQ) Catalog # reactions
Absolute Human Telomere Length and Mitochondrial DNA Copy Number Dual Quantification qpcr Assay Kit (AHDQ) Catalog #8958 100 reactions Product Description Telomeres are repetitive nucleotide elements at
More informationThe Biotechnology Toolbox
Chapter 15 The Biotechnology Toolbox Cutting and Pasting DNA Cutting DNA Restriction endonuclease or restriction enzymes Cellular protection mechanism for infected foreign DNA Recognition and cutting specific
More informationTHUNDERBIRD SYBR qpcr Mix
Instruction manual THUNDERBIRD SYBR qpcr Mix 1304 A4251K THUNDERBIRD SYBR qpcr Mix QPS-201T 1 ml x 1 QPS-201 1.67 ml x 3 Contents [1] Introduction [2] Components [3] Primer design [4] Template DNA [5]
More informationEpiQuik Tissue Methyl-CpG Binding Domain Protein 2 ChIP Kit
EpiQuik Tissue Methyl-CpG Binding Domain Protein 2 ChIP Kit Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Tissue Methyl-CpG Binding Domain Protein 2 ChIP Kit is suitable for
More informationBRCA MAQ USER GUIDE Version 1.0
BRCA MAQ USER GUIDE Version 1.0 For Copy Number Analysis Assays IFU604 v170797 www.multiplicom.com 2012 Multiplicom NV, all rights reserved Table of Contents Introduction to Multiplex Amplicon Quantification...2
More informationSample to Insight. Dr. Bhagyashree S. Birla NGS Field Application Scientist
Dr. Bhagyashree S. Birla NGS Field Application Scientist bhagyashree.birla@qiagen.com NGS spans a broad range of applications DNA Applications Human ID Liquid biopsy Biomarker discovery Inherited and somatic
More informationAutomation of Lexogen s QuantSeq 3 mrna-seq Library Prep Kits on the Biomek FX p NGS Workstation
Automation of Lexogen s QuantSeq 3 mrna-seq Library Prep Kits on the Biomek FX p NGS Workstation The Lexogen QuantSeq 3 mrna-seq Library Prep Kits for Illumina (FWD and REV) produce ready-to-sequence libraries
More informationAmpFlSTR Identifiler PCR Amplification Kit
Product Bulletin Human Identification AmpFlSTR Identifiler PCR Amplification Kit Fifteen STR (short tandem repeat) loci and Amelogenin co-amplified in a single tube Incorporation of the same proven, reliable
More informationSupplemental Table S1. Experimental details of the qpcr analyses according to the checklist of the MIQE* guidelines.
Supplemental Table S1, page 1 Supplemental Table S1. Experimental details of the qpcr analyses according to the checklist of the MIQE* guidelines. ITEM TO ERXPERIMENTAL DESIGN Definition of experimental
More informationGenetic Technologies
Genetic Technologies Distinguish the terms biotechnology, recombinant DNA technology, transgenic organisms, genetic engineering Understand the two basic techniques to obtain selective fragments of DNA
More informationComplete protocol in 110 minutes Enzymatic fragmentation without sonication One-step fragmentation/tagging to save time
Molecular Cloning Laboratories Manual Version 1.2 Product name: MCNext UT DNA Sample Prep Kit Cat #: MCUDS-4, MCUDS-24, MCUDS-96 Description: This protocol explains how to prepare up to 96 pooled indexed
More informationEPIGENTEK. EpiQuik Tissue Chromatin Immunoprecipitation Kit. Base Catalog # P-2003 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE
EpiQuik Tissue Chromatin Immunoprecipitation Kit Base Catalog # P-2003 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Tissue Chromatin Immunoprecipitation Kit is suitable for combining the specificity
More informationQuant One Step RT-PCR Kit
1. Quant One Step RT-PCR Kit For fast and sensitive one-step RT-PCR www.tiangen.com/en RT121221 Quant One Step RT-PCR Kit Kit Contents Cat. no. KR113 Contents Hotmaster Taq Polymerase (2.5 U/μl) Quant
More informationLecture #1. Introduction to microarray technology
Lecture #1 Introduction to microarray technology Outline General purpose Microarray assay concept Basic microarray experimental process cdna/two channel arrays Oligonucleotide arrays Exon arrays Comparing
More informationIsoFluxTM. System. The next generation of CTC Analysis is here
IsoFluxTM System The next generation of CTC Analysis is here Product Overview IsoFlux System The next generation of circulating tumor cell analysis is here The IsoFlux System enriches intact rare cells
More informationMethods in virus diagnosis PCR techniques
Methods in virus diagnosis PCR techniques 450 MBIO PRACTICAL LESSON 5 Molecular Methods Methods based on the detection of viral genome are also commonly known as molecular methods. It is often said that
More informationSuperTCRExpress TM Human TCR Vβ Repertoire CDR3 Diversity Determination (Spectratyping) and Quantitative Analysis Kit
SuperTCRExpress TM Human TCR Vβ Repertoire CDR3 Diversity Determination (Spectratyping) and Quantitative Analysis Kit Cat. No. H0521 Size: 2 sets (22 Vβ families/each, with enzymes) H0522 Size: 4 sets
More informationMassARRAY Genetic Analysis System. Genotyping Methylation Analysis Molecular Typing Somatic Mutation Profiling Quantitative Gene Expression (QGE)
MassARRAY Genetic Analysis System Genotyping Methylation Analysis Molecular Typing Somatic Mutation Profiling Quantitative Gene Expression (QGE) MassARRAY Genetic Analysis System * Overview Next-generation
More informationqpcr Quantitative PCR or Real-time PCR Gives a measurement of PCR product at end of each cycle real time
qpcr qpcr Quantitative PCR or Real-time PCR Gives a measurement of PCR product at end of each cycle real time Differs from endpoint PCR gel on last cycle Used to determines relative amount of template
More informationQuantitative Telomerase Detection Kit (QTD Kit)
Quantitative Telomerase Detection Kit (QTD Kit) Catalog No. MT3010, MT3011, MT3012 For Research Use Only. Not for use in diagnostic procedures 1 Table of Contents 1. Introduction Background Product Overview
More informationEpiQuik Methyl-CpG Binding Domain Protein 2 ChIP Kit
EpiQuik Methyl-CpG Binding Domain Protein 2 ChIP Kit Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Methyl-CpG Binding Domain Protein 2 ChIP Kit is suitable for combining the
More informationP HENIX. PHENIX PCR Enzyme Guide Tools For Life Science Discovery RESEARCH PRODUCTS
PHENIX PCR Enzyme Guide PHENIX offers a broad line of premium quality PCR Enzymes. This PCR Enzyme Guide will help simplify your polymerase selection process. Each DNA Polymerase has different characteristics
More informationRapid Method for the Purification of Total RNA from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Samples
Application Note 17 RNA Sample Preparation Rapid Method for the Purification of Total RNA from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Samples M. Melmogy 1, V. Misic 1, B. Lam, PhD 1, C. Dobbin,
More informationRapid Method for the Purification of Total RNA from Formalin- Fixed Paraffin-Embedded (FFPE) Tissue Samples
Application Note 17 RNA Sample Preparation Rapid Method for the Purification of Total RNA from Formalin- Fixed Paraffin-Embedded (FFPE) Tissue Samples M. Melmogy 1, V. Misic 1, B. Lam, PhD 1, C. Dobbin,
More informationWhat is DNA? Deoxyribonucleic Acid The inherited genetic material that makes us what we are
DNA Basic Genetics What is DNA? DNA is Deoxyribonucleic Acid The inherited genetic material that makes us what we are DNA in the Cell Human Genome ~3 billion base pairs of DNA 30,000-35,000 genes Population-each
More informationDNA/RNA MICROARRAYS NOTE: USE THIS KIT WITHIN 6 MONTHS OF RECEIPT.
DNA/RNA MICROARRAYS This protocol is based on the EDVOTEK protocol DNA/RNA Microarrays. 10 groups of students NOTE: USE THIS KIT WITHIN 6 MONTHS OF RECEIPT. 1. EXPERIMENT OBJECTIVE The objective of this
More informationFunctional DNA Quality Analysis Improves the Accuracy of Next Generation Sequencing from Clinical Specimens
Functional DNA Quality Analysis Improves the Accuracy of Next Generation Sequencing from Clinical Specimens Overview We have developed a novel QC, the SuraSeq DNA Quantitative Functional Index (QFI ).
More informationNon-Organic-Based Isolation of Mammalian microrna using Norgen s microrna Purification Kit
Application Note 13 RNA Sample Preparation Non-Organic-Based Isolation of Mammalian microrna using Norgen s microrna Purification Kit B. Lam, PhD 1, P. Roberts, MSc 1 Y. Haj-Ahmad, M.Sc., Ph.D 1,2 1 Norgen
More informationPV92 PCR Bio Informatics
Purpose of PCR Chromosome 16 PV92 PV92 PCR Bio Informatics Alu insert, PV92 locus, chromosome 16 Introduce the polymerase chain reaction (PCR) technique Apply PCR to population genetics Directly measure
More informationRapid, Accurate and Flexible DNA Quantitation Using the QuantiFluor dsdna System on the MANTIS Liquid Handler
Rapid, Accurate and Flexible DNA Quantitation Using the QuantiFluor dsdna System on the MANTIS Liquid Handler Materials Required MANTIS Instrument (Formulatrix) High Volume Chips (Formulatrix Cat.# MCHS6)
More informationDNA METHYLATION NH 2 H 3. Solutions using bisulfite conversion and immunocapture Ideal for NGS, Sanger sequencing, Pyrosequencing, and qpcr
NH 2 DNA METHYLATION H 3 C Solutions using bisulfite conversion and immunocapture Ideal for NGS, Sanger sequencing, Pyrosequencing, and qpcr NH 2 N N H PAGE 3 Understanding DNA Methylation DNA methylation
More informationDNA Microarray Technology
CHAPTER 1 DNA Microarray Technology All living organisms are composed of cells. As a functional unit, each cell can make copies of itself, and this process depends on a proper replication of the genetic
More informationAssays for gene expression and protein production
Assays for gene expression and protein production Module 3, Lecture 5! 20.109 Spring 2011! Topics for Lecture 5 Measuring protein levels! Measuring transcript levels! Imaging assays! 2 Module overview:
More informationReverse Transcription & RT-PCR
Creating Gene Expression Solutions Reverse Transcription & RT-PCR Reverse transcription, a process that involves a reverse transcriptase (RTase) which uses RNA as the template to make complementary DNA
More informationSMARTer Ultra Low RNA Kit for Illumina Sequencing Two powerful technologies combine to enable sequencing with ultra-low levels of RNA
SMARTer Ultra Low RNA Kit for Illumina Sequencing Two powerful technologies combine to enable sequencing with ultra-low levels of RNA The most sensitive cdna synthesis technology, combined with next-generation
More informationEPIGENTEK. EpiQuik Methylated DNA Immunoprecipitation Kit. Base Catalog # P-2019 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE
EpiQuik Methylated DNA Immunoprecipitation Kit Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik MeDIP Kit can be used for immunoprecipitating the methylated DNA from a broad range
More informationRelative Mouse Telomere Length Quantification qpcr Assay Kit (RMTLQ) Catalog #M reactions
Relative Mouse Telomere Length Quantification qpcr Assay Kit (RMTLQ) Catalog #M8908 100 reactions Product Description Telomeres are repetitive nucleotide elements at the ends of chromosomes that protect
More informationLiquidBiopsy LIQUIDBIOPSY. Automated Rare Template Isolation Platform
LiquidBiopsy LIQUIDBIOPSY Automated Rare Template Isolation Platform Enabling cancer research with highly multiplexed molecular analysis of serially collected blood samples The LiquidBiopsy Platform simplifies
More informationTAKE YOUR NEXT STEP WITH CONFIDENCE
TAKE YOUR NEXT STEP WITH CONFIDENCE Biomek-Automated Genomic Solutions IT STARTS WITH DEFINING THE RIGHT WORKFLOW SOLUTION FOR YOU Human Health Agricultural Animal Health Environmental Food & Beverage
More informationTo NanoDrop or Not to NanoDrop : Choosing the Most Appropriate Method for Nucleic Acid Quantitation
To NanoDrop or Not to NanoDrop : Choosing the Most Appropriate Method for Nucleic Acid Quantitation Presented by: Doug Wieczorek, Ph.D. Senior Application Scientist Presentation Outline Nucleic acid quantitation
More informationMarketplace. Essentials Tour The bright side of PCR. molecular biology. thermofisher.com/marketplace
molecular biology Marketplace The bright side of PCR Special offers on Thermo Scientific products: Agarose powder DNA ladders Electrophoresis dyes Enhanced Taq DNA polymerases High-fidelity DNA polymerases
More informationHigh Throughput Sequencing the Multi-Tool of Life Sciences. Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center
High Throughput Sequencing the Multi-Tool of Life Sciences Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center Complementary Approaches Illumina Still-imaging of clusters (~1000
More informationAPPLICATION NOTE. The QuantStudio 12K Flex Real-Time PCR System
APPLICATION NOTE The QuantStudio 12K Flex Real-Time PCR System Streamline high-throughput gene expression studies with SuperScript III Platinum One-Step Quantitative RT-PCR and OpenArray technology on
More informationEmbrace the Future of Electrophoresis
Embrace the Future of Electrophoresis Analysis of 12 samples in as little as 3 minutes Unattended analysis of up to 96 samples Resolution down to 3 5 bp for fragments
More informationIsolation of total nucleic acids from FFPE tissues using FormaPure DNA
APPLICATION NOTE Isolation of total nucleic acids from FFPE tissues using FormaPure DNA Jung Hoon Doh, Ph.D. Senior Application Scientist Beckman Coulter Life Sciences, Indianapolis, IN USA Summary Extensive
More informationChapter 10 Genetic Engineering: A Revolution in Molecular Biology
Chapter 10 Genetic Engineering: A Revolution in Molecular Biology Genetic Engineering Direct, deliberate modification of an organism s genome bioengineering Biotechnology use of an organism s biochemical
More informationB. Incorrect! Ligation is also a necessary step for cloning.
Genetics - Problem Drill 15: The Techniques in Molecular Genetics No. 1 of 10 1. Which of the following is not part of the normal process of cloning recombinant DNA in bacteria? (A) Restriction endonuclease
More informationTarget Enrichment Strategies for Next Generation Sequencing
Target Enrichment Strategies for Next Generation Sequencing Anuj Gupta, PhD Agilent Technologies, New Delhi Genotypic Conference, Sept 2014 NGS Timeline Information burst Nearly 30,000 human genomes sequenced
More informationMutations during meiosis and germ line division lead to genetic variation between individuals
Mutations during meiosis and germ line division lead to genetic variation between individuals Types of mutations: point mutations indels (insertion/deletion) copy number variation structural rearrangements
More informationDeveloping an Accurate and Precise Companion Diagnostic Assay for Targeted Therapies in DLBCL
Developing an Accurate and Precise Companion Diagnostic Assay for Targeted Therapies in DLBCL James Storhoff, Ph.D. Senior Manager, Diagnostic Test Development World Cdx, Boston, Sep. 10th Molecules That
More informationTECH NOTE SMARTer T-cell receptor profiling in single cells
TECH NOTE SMARTer T-cell receptor profiling in single cells Flexible workflow: Illumina-ready libraries from FACS or manually sorted single cells Ease of use: Optimized indexing allows for pooling 96 cells
More informationEPIGENTEK. EpiQuik Chromatin Immunoprecipitation Kit. Base Catalog # P-2002 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE
EpiQuik Chromatin Immunoprecipitation Kit Base Catalog # P-2002 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Chromatin Immunoprecipitation Kit is suitable for combining the specificity of
More informationSimple, Complete Workflows for Gene Expression Analysis without RNA Purification
Product Bulletin SYBR Green Cells-to-Ct Kits SYBR Green Cells-to-Ct Kits Simple, Complete Workflows for Gene Expression Analysis without RNA Purification 7 10 min total 5x 5' 5' 3' 3' 3' mrna 5' RT primer
More informationLATE-PCR. Linear-After-The-Exponential
LATE-PCR Linear-After-The-Exponential A Patented Invention of the Laboratory of Human Genetics and Reproductive Biology Lab. Director: Lawrence J. Wangh, Ph.D. Department of Biology, Brandeis University,
More informationMassARRAY. Quantitative Methylation Analysis. High Resolution Profiling. Simplified with EpiTYPER.
MassARRAY Quantitative Methylation Analysis High Resolution Profiling. Simplified with EpiTYPER. MassARRAY Quantitative Methylation Analysis Overview Unprecedented Levels of Performance The EpiTYPER assay
More informationSEQUENCING FROM SAMPLE TO SEQUENCE READY
SEQUENCING FROM SAMPLE TO SEQUENCE READY ACCESS ARRAY SYSTEM HIGH-QUALITY LIBRARIES NOT ONCE, BUT EVERY TIME n The highest quality amplicons more sensitive, accurate, and specific n Full support for all
More informationWet-lab Considerations for Illumina data analysis
Wet-lab Considerations for Illumina data analysis Based on a presentation by Henriette O Geen Lutz Froenicke DNA Technologies and Expression Analysis Cores UCD Genome Center Complementary Approaches Illumina
More informationImpact of gdna Integrity on the Outcome of DNA Methylation Studies
Impact of gdna Integrity on the Outcome of DNA Methylation Studies Application Note Nucleic Acid Analysis Authors Emily Putnam, Keith Booher, and Xueguang Sun Zymo Research Corporation, Irvine, CA, USA
More informationPersonalized Genetic Technology and Novel Multiplex Quantitative PCR
Personalized Genetic Technology and Novel Multiplex Quantitative PCR Hong Kong University Tiffany Jiang Ph.D. Asia Pacific Marketing Manager Beckman Coulter At your fingertip!! Multiplex Gene Expression
More informationBIOLOGY Dr.Locke Lecture# 27 An Introduction to Polymerase Chain Reaction (PCR)
BIOLOGY 207 - Dr.Locke Lecture# 27 An Introduction to Polymerase Chain Reaction (PCR) Required readings and problems: Reading: Open Genetics, Chapter 8.1 Problems: Chapter 8 Optional Griffiths (2008) 9
More informationDEPArray Technology. Sorting and Recovery of Rare Cells
DEPArray Technology Sorting and Recovery of Rare Cells Delivering pure, single, viable cells The DEPArray system from Silicon Biosystems is the only automated instrument that can identify, quantify, and
More informationAccess Array BRCA1 / BRCA2 / TP53 Target-Specific Panel Build the highest quality amplicon libraries with qualified assays
DATA SHEET PN 100-3489 B1 Access Array BRCA1 / BRCA2 / TP53 Target-Specific Panel Build the highest quality amplicon libraries with qualified assays Covers 100% of the exons within the genes Supported
More informationPrimePCR Assay Validation Report
Gene Information Gene Name minichromosome maintenance complex component 8 Gene Symbol Organism Gene Summary Gene Aliases RefSeq Accession No. UniGene ID Ensembl Gene ID MCM8 Human The protein encoded by
More informationLecture Four. Molecular Approaches I: Nucleic Acids
Lecture Four. Molecular Approaches I: Nucleic Acids I. Recombinant DNA and Gene Cloning Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single
More informationEPIGENTEK. ChromaFlash Chromatin Extraction Kit. Base Catalog # P-2001 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE KIT CONTENTS
ChromaFlash Chromatin Extraction Kit Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses: The ChromaFlash Chromatin Extraction Kit is suitable for isolating chromatin or DNA-protein complex
More informationExecutive Summary. clinical supply services
clinical supply services case study Development and NDA-level validation of quantitative polymerase chain reaction (qpcr) procedure for detection and quantification of residual E.coli genomic DNA Executive
More informationUtility of Branched DNA Hybridization Methodology for the Quantitation of Oligonucleotides
Utility of Branched DNA Hybridization Methodology for the Quantitation of Oligonucleotides Laboratory Sciences, MPI Research, A Charles River Company Amy Smith, BA, Senior Director, Bioanalytical/Analytical
More informationQIAseq SPE technology for Illumina : Redefining amplicon sequencing
Application Note QIAseq SPE technology for Illumina : Redefining amplicon sequencing Amplicon-based enrichment and sequencing takes advantage of PCR workflows to turn amplicons that represent regions of
More informationHaloPlex HS. Get to Know Your DNA. Every Single Fragment. Kevin Poon, Ph.D.
HaloPlex HS Get to Know Your DNA. Every Single Fragment. Kevin Poon, Ph.D. Sr. Global Product Manager Diagnostics & Genomics Group Agilent Technologies For Research Use Only. Not for Use in Diagnostic
More informationSALSA MLPA probemix P125-B1 Mitochondria Lot B1-0216, B1-0312, B
SALSA MLPA probemix P125-B1 Mitochondria Lot B1-0216, B1-0312, B1-0609. Mitochondrial DNA (mtdna) differs from nuclear DNA in several ways. The complete mitochondrial genome is circular and only 16,569
More information