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.