Assay

Company Confidential

Part 1: You Say, Assay

Part 2: PCR- Poly What?


Who cares?


Did you say “essay?”

Definition: Analysis (n), analyze (v)


More precision please.

• Determine presence of sample

• E.g. does the plasmid contain my gene?

• Determine quantity of sample

• E.g. how much protein do I have?

• Determine activity of drug or biochemical in a sample

• E.g. what’s the activity of my protein?


Assays @ Work: Protein Purification

Protocol• Transfect bacteria with plasmid containing your DNA, plate on to agar

• Determine optical density of cells

• Pick bacteria colony from agar plate expressing your protein

• Confirm bacteria contains your sequence

• Grow bacteria expressing your protein in broth overnight

• Determine optical density of cells

• Centrifuge cells, remove broth, add buffer

• Break open cells

• Determine protein concentration

• Determine protein activity

• Determine protein purity

• Fractionate protein with a salt




• Chromatography steps to enrich your protein




• Confirm protein identity


Wide world of assays

What assay am I?


Does it work?

• Accuracy - does it give the right answer?

• Precision - does it give a consistent answer?

• Reliability – does it work every time?

• Sensitivity -how little analyte can you detect?

• Dynamic range – can you measure your analyte over a wide concentration range?

• Robustness – will the assay work under non-optimal conditions?


Practically speaking….

• Speed (“time-to-result”)

• Throughput

• Automation

• Ease-of-use

• Workflow

• Cost

• Size (“footprint”)


Last word: Does it smell?


Questions?


PCR- Poly What?


Polymerase Chain Reaction

• In short: it is a method used to analyze a

short sequence of DNA or RNA

• PCR is used to reproduce (amplify)

selected sections of DNA or RNA

• The basics of what’s needed:

• Thermal cycler

• Two primers

• An enzyme called polymerase

• A pile of DNA building blocks that the

polymerase needs to make that copy


Some of the Applications of PCR

• Diagnostics

• Forensic analysis

• Detection of infectious agents

• Rare mutation detection

• Gene expression

• DNA sequencing

• Copy number variation

• SNP genotyping


So Fancy


3 Steps to PCR

• Denaturation

• The double strand melts open to single stranded DNA

• Annealing

• The primers pair up (anneal) with the single-stranded "template" (The template is the

sequence of DNA to be copied.)

• On the small length of double-stranded DNA (the joined primer and template), the polymerase

attaches and starts copying the template.

• Extension

• The polymerase works synthesizing making a new DNA strand

RINSE. LATHER. REPEAT.

http://bit.ly/xGvqec


Real-Time PCR

• Real-Time PCR (qPCR)

• Enables both detection and quantification

• Measures PCR amplification as it occurs– hence the name “real-time”

• Uses fluorescent probes

• Requires the comparison of an unknown to a standard to obtain quantitative information

• Analogue measurement based on measuring amplification after each cycle of PCR


It’s time to digitize

• Digital PCR

• Works like real-time PCR, except it first partitions the sample into hundreds or even thousands

of reaction chambers (chips, plates, droplets)

• Some may have template, others may not

• Then amplification occurs with qPCR

• Reactions with target are read as a “1” and those without as a “0”

• Together with the volume of each reaction and the total number of reactions analyzed, an

estimate of the absolute target DNA concentration is calculated

• Droplet Digital PCR

• Water-in-oil droplets (approximately 20,000)

• Gives off fluorescent + or – signals

• Software calculates the concentration of target DNA


Real-time vs. Digital

Assay

Technology

Transcript of Assay