LIFE SCIENCE TECHNOLOGIES:The Digital PCR Revolution DNA
(Nucleotide) (, deoxyribose)(phosphate group)(AGCTU)
DNARNA
PCR1.(primary structure)2. (secondary structure)(-helix)(beta sheet)3. (tertiary structure)4. (quaternary structure)
(primary structure)C=O (secondary structure)(peptide bond): 1. - 2. -
(tertiary structure)domain
(quaternary structure)()
DNAdeletion-DNAinversion-translocationduplication
contmRNA-(synapses)
cont--A-X-X-21
(conformational rearrangement) (misfolding)(self-association)(-linkage)(tissue deposition)(inclusion body) (Alzheimers disease)(Parkinsons disease)(Bovine spongiform encephalopathy
DNA hydrogen bondings
Primer: A short segment of DNA sequences
Examples:
Primer 1: ATTGC
Primer 2: GGTGCAPrimer and DNA binding
Examples:
Primer 1: ATTGC DNA 1 : TAACGTCGATGCCTTAG
Primer 2: GGTGCADNA 2 : CCACGTTATCCGTAGCGTC Primer and DNA un-binding
Primer 1: ATTGC DNA 1 : TAACGTCGATGCCTTAG3(A-T) pairs and 2(G-C) pairs:12 hydrogen bondings
Primer 2: GGTGCADNA 2 : CCACGTTATCCGTAGCGTC2(A-T) pairs and 4(G-C) pairs:16 hydrogen bondings
PCR: Polymerase chain reaction
PCR
PCRReal-Time PCRReal-time PCRFluorescent reporter probe method
Real-time PCR due to variations in reaction kinetics,different quantities of PCR product by the plateau phase of the reaction it will be more precise to take measurements during the exponential phase
AdvantageReduce the experiment timemore sensitive & accurate
Ct value The PCR cycle at which the sample reaches a fluorescent intensity above background is the Cycle Threshold or Ct making it possible to determine the starting concentration of nucleic acid
Digital PCRPresent By Bert Vogelstein, Kenneth W. Kinzler Proc Natil.Acad. Sci. 9236-9241. 1999IntroductionCancerGenecell proliferationcancer, Curable (minor tissue injury) curable(major tissue injury)Non-curable. VirusHIV (AIDS) with treatment Difficult to detect HIV virus.Monitor virus mutation and latent virusWhy we need to develop Digital PCR???Detection small tumor cell from large amount of normal cell (Looking a needle in the haystack)UrineStoolBloodNot contamination by normal cell after amplificationAbsolute quantification
Digital PCR method
Special probe designStem-loop formationTemperature change shape changeFlourescence energy was inversely proportional to 6 power of distance between two point.
Probe and target DNAMB-Green attach to Mutation site DNAMB-Red attach to normal siteSuccessful amplification Wild type PCR both MB-Red and MB-Green gives lightSuccessful amplification of Mutation type PCR on ly MB-Red gives light
Dilute and PCR amplificationTry to Dilute DNA to copy per wellRepeat temperatrure control PCR amplification Probe detectionRead the data (Red/Green ratio)
C-Ki-Ras mutation and R/G ratio
C-Ki-Ras from tumor cell
Stool sample for C-Ki-Ras (Colorectal cancer)
Characteristics of Digital PCRPrevent contaminationDilution and Partition to ultra small moleculeIn situ PCR amplification in each well Absolute quantificationSimply detection of Mutation/Wild type, not every Mutation typePoisson distributionQuality controlThermal cycleSpecial primer design
Video DemonstrationIntroduction o Digital PCRThank you
Comparison between Traditional PCR and Real-time PCR and Digital PCR
Three phases of PCR
Detection areaCycle threshold of real-time PCR
Digital PCR works by partitioning a sample into many individual real-time PCR reactions, some portion of these reactions contain the target molecules(positive) while others do not (negative).The fraction of negative answers is used as reference.
