Digital droplet pcr.pptx new technology of pcr

Digita pcr subtype

1. Digital droplet pcr

2. PolymeRase chain reaction It is a technique that allows rapid amplification of a specific segment of DNA. It makes billions of copies of a specific DNA Fragment ,which allows detection and identification of gene sequences TYPES OF PCR: Real time PCR Nested PCR Multiplex PCR Quantitative PCR

3. TRADITIONAL OVER DIGITAL PCR Digital PCR ( dPCR ) enables precise, highly sensitive quantification of nucleic acids.   Traditional PCR  is an end-point analysis that is semi-quantitative because the amplified product is detected by agarose gel electrophoresis.   Real-time PCR (or qPCR)  uses  fluorescence-based detection  to allow the measurement of accumulated amplified product as the reaction progresses. qPCR requires normalization to controls (either to a reference or to a standard curve), allowing only relative quantification.

4. ddpcr Digital PCR ( dPCR ) is a highly precise and sensitive method of quantitative PCR. A technique used in molecular biology to amplify and quantify DNA. The key distinction of dPCR compared to traditional quantitative PCR (qPCR) is the way it partitions the DNA sample. It is a type of PCR approach that partitons a sample into thousands of individual droplets, allowing for absolute quantification of target nucleic acids.

5. It is a method for performing digital PCR that is based on water –oil emulsion droplet technology. A sample is fractioned into 20,000 droplets and PCR amplification of the template molecules occur in each individual droplet. Droplets are formed in a water oil emulsion to form the partitions that separate the template DNA molecules

6. Benefits : This technique has a smaller sample requirement than other Digital PCR systems, reducing cost and precious samples Detect rare DNA target copies with unmatched sensitivity Measure gene expression levels with accuracy Determine copy number variation with accuracy

7. PRINCIPLE ddPCR technology uses a combination of microfluidis and proprietary surfactant chemistries to divide PCR samples into water-in-oil droplets. The droplets support PCR amplification of the template molecules and use reagents and workflows similar to those used for most standard TaqMan probe-based assays. Following PCR, each droplet is analyzed or read in a flow cytometer to determine the fraction of PCR-positive droplets in the original sample. These data are then analyzed using Poisson statistics to determine the target DNA template concentration in the original sample.

8. workflow Combining DNA sample and primers and probes with Supermix to create 8 prepared sample. 2. Sample will be load by 20 μl in to each individual wells of eight channel disposable droplet generator cartridge

9. 1. Droplet Generation: Load the cartridge into Droplet Generator, to create an emulsion of 20,000 nanoliter-sized droplets ready for PCR for each of the 8 prerpared samples The droplets created by the Droplet Generator should be uniform in size and volume

10. In during Droplet Generation a specified material is added to stabilize the droplets

11. Seal the PCR plate with pcr plate sealer

12. 3.DROPLET READING After PCR amplification of the nucleic acid target in the droplets, the samples are placed in the Droplet Reader. • Reader analyzes each droplet individually using a two-color detection system (set to detect FAM and either HEX or VIC), enabling multiplexed analysis for different targets in the same sample. • The droplet reader and software count the PCR-positive and PCR -Negative droplets. • Fluorescence measurements for each droplet in two optical channels are used to count the numbers of positive and negative droplets per sample

13. In ddPCR , the QuantaSoft software measures the numbers of droplets that are positive and negative for each fluorophore (for example, FAM and HEX ) In a sample

14. Each droplet in a sample is plotted on a graph of fluorescence intensity versus droplet number. All positive droplets (those above the threshold intensity indicated by the red line) are scored as positive, and each is assigned a value of 1. All negative droplets (those below the threshold) are scored as negative, and each is assigned a value of 0 (zero). This counting technique provides a digital signal from which to calculate the starting target DNA concentration by a statistical analysis of the numbers of positive and negative droplets in a given sample. 4. ddPCR Data Analysis

15. Advantages of ddPCR Absolute quantification — ddPCR provides a absolute count of target DNA copies per input sample without the need for running standard curves, making this technique ideal for target DNA measurements, viral load analysis, and microbial quantification. 2. Genomic alterations such as gene copy number variation (CNV) — CNVs result in too variability, complex behavioral traits, and disease. ddPCR enables measurement of 1.2x differences in gene copy number.

16. unparalleled precision  — the massive sample partitioning afforded by ddPCR enables the reliable measurement of small fold differences in target DNA sequence copy numbers among samples Increased signal-to-noise ratio  — high-copy templates and background are diluted, effectively enriching template concentration in target-positive partitions, allowing for the sensitive detection of rare targets and enabling a ±10% precision in quantification Removal of PCR bias  — error rates are reduced by removing the amplification efficiency reliance of qPCR, enabling the detection of small (1.2-fold) differences

17. Simplified quantification  — neither calibration standards nor a reference (the  ΔΔCq  method) is required for absolute quantification Reduced consumable costs  — reaction volumes are in the pico- to nanoliter ranges, reducing reagent use and the sample quantity required for each data point Lower equipment costs  — the emulsion-based reaction system means that the PCR reactions can be performed in a standard thermal cycler without complex chips or microfluidics Superior partitioning  — ddPCR technology yields 20,000 droplets per 20 µl sample, nearly two million partitioned PCR reactions in a 96-well plate, whereas chip-based digital PCR systems produce only hundreds or thousands of partitions. The greater number of partitions yields higher accuracy

18. Detection of rare sequences — researchers must amplify single genes in a complex sample, such as a few tumor cells in a wild-type background. ddPCR is sensitive enough to detect rare mutations or sequences Gene expression and microRNA analysis — ddPCR provides stand-alone absolute quantification of expression levels, especially low-abundance microRNAs, with sensitivity and precision

19. Next-generation sequencing (NGS) — ddPCR quantifies NGS sample library preparations to increase sequencing accuracy and reduce run repeats. Validate sequencing results such as single nucleotide polymorphisms or copy number variations with absolute quantification Single cell analysis — the high degree (10- to 100-fold) of cell-cell variation in gene expression and genomic content among homogeneous post-mitotic, progenitor, and stem cell populations drives a need for analysis from single cells. ddPCR enables low copy number quantification

20. Serial dilution is laborious and introduces the possiblity of pipetting error; competing chip-based systems rely on complex fluidics schemes for partitioning. Droplet Digital PCR addresses these shortcomings by massively partitioning the sample in the fluid phase in one step. The creation of tens of thousands of droplets means that a single sample can generate tens of thousands of data points rather than a single result, bringing the power of statistical analysis inherent in digital PCR into practical application. Bio-Rad's Droplet Digital PCR System automates the ddPCR workflow of droplet generation, thermal cycling, droplet reading, and data analysis, making this technology accessible to the working research laboratory.