Biotechnology course

Dr. Stella Georgiou

sgeorgiou@bioacademy.gr

Polymerase Chain Reaction (PCR)

Overview

• What biological question is to be answered?

What is the goal of the experiment?

• What is the total number of genes to be analyzed?

• What control samples and genes will be used to measure the changes

in expression levels?

• Are there any limitations to the amount of target material available?

• What is the sensitivity required to obtain the data necessary to

answer?

from DNA to RNA

Gene expression changes between different cell types, developmental changes,

treatments, diseases/infections/cancer, stress, alternative splicing, etc…

Reverse transcription:

When and why to check for RNA expression?

what usually happens in the lab..

PCR relies on the complementary base pairing of the DNA

How everything starts..

What really happens..

PCR experiment

PCR steps

RV

FW Fold expression= 2n

n = number of cycles

Start from the basics

PC

R p

rod

uct

Cycle number

PCR kinetics

products of:

Endpoint PCRDigital PCR

Microfluidic PCR

Theoretical doubling

of the starting

amount in each cycle

• Primers…?

Successful PCR: everything is optimized

MW marker• Template DNA: genomic DNA (gDNA), complementary DNA (cDNA), and plasmid DNA,

e.g. 5–50 ng of gDNA

• DNA polymerase: in a typical 50 µL reaction, 1–2 units of DNA polymerase

• Deoxynucleoside triphosphates (dNTPs): dATP, dCTP, dGTP, and dTTP are

in excess. Final concentration of each dNTP is generally 0.2 mM

• Buffer: suitable chemical environment for activity of DNA polymerase. Buffer pH 8.0-9.5

• Mg2+, other cofactors: Mg2+ functions as a cofactor for activity of DNA polymerases

https://www.addgene.org/protocols/primer-design/

How to choose FW & RV primers?

Primers

Important for the analysis

Gene-specific!Or transcript-specific

PCR applications

Who’s the father?

real-time QPCR measures the fluorescence at each cycle as the

amplification progresses

quantification of the template is based on the fluorescent signal

during the exponential phase of amplification

How to monitor the levels in gene expression?

principles of real-time pcr: amplification plot

Ct: The first cycle at which the

instrument can distinguish the

amplification generated fluorescence as

being above the ambient background

The greater the amount of initial DNA

template in the sample, the earlier the Ct

value for that sample

SYBR-Green chemistry:• cost—effective• easy to use

Important for the analysis: Sequences of primers or probes should be removed!

Probe-Based chemistries:• higher detection specificity• multiple probes labeled with different reporter

dyes allow detection of more than one target in a single reaction (multiplex QPCR)

How can the products be spotted?

The fluorescence intensity increases proportionally

with each amplification cycle in response to the

increased amplicon concentration

PCR products visualization

Old times:Ethidium bromide

Cost-effective

mutagenic

PCR products visualization

Modern times:Non-toxic dyes

GelRed®

SYBR Safe ®

principles of real-time pcr: dissociation curve

1 amplified product

2 amplified products

principles of real-time pcr: dissociation curve

1 amplified product

2 amplified products

How many amplified products?

Comparative or Relative quantitation:

comparing the levels of a target gene in test

samples relative to a sample of reference

Quantitative as Absolute quantitation:

a standard curve to quantitate the amount of target present

in an Unknown sample

or a series of Standard samples, containing dilutions of a

known amount of target

The standard curve is then used to derive the initial template

quantity in Unknown wells based on their Ct values

Do I need a standard curve?

Since there is a theoretical doubling of the starting amount in each cycle what is the efficiency? E ̴̴̴̴̴̴̴̴̴̴̴̴̴̴̴̴ 2

o 18S rRNA (18S ribosomal RNA),

o 28S rRNA (28S ribosomal RNA),

o TUBA (α-tubulin),

o ACTB (β-actin),

o EF1A (elongation factor 1α),

o GAPDH (glyceraldehyde-3-phosphate dehydrogenase),

o HPRT (hypoxanthine phosphoribosyl transferase)

housekeeping or reference genes

Comparative or Relative quantitation:

comparing the levels of a target gene in test

samples relative to a sample of reference

Relative quantitation: we need reference genes

Important for the analysis: Include a negative control & a positive control

PCR normalization

PCR controls

microfluidics

The principle

When a particle passes through a laser beam, it scatters light according to its

physical properties.

e.g. A larger particle will cause higher deviation of the light. In case this particle is also

fluorescent, after being excited with light of the appropriate wavelength, it will emit light at

another (usually higher) wavelength.

An optical-to-electronic coupling system is used to record how each cell

scatters laser light and emits fluorescence.

For cells

Cells should be in single-cell suspension

Cells suspended in a liquid (fluidics system) pass through a laser beam causing

light scatter that can be sensed by appropriate detectors (optics system).

The electronics system process the signal from the detectors giving numbers that

represent the features (size and granularity) of the cells. Jalali, M., Saldanha, F. Y. L., & Jalali, M. (Eds.). (2017). Basic

science methods for clinical researchers. London: Academic Press.

microfluidics benefits: large number of samples, speed, parallelization, sensitivity, multiplexity,

reduction of reagents cost

Microfluidics have been used for PCR, qPCR, RT-PCR

microfluidic PCR

microfluidics drawbacks: PCR inhibition and carryover contamination

Zhang Y, Jiang HR. Anal Chim Acta. 2016 Mar 31;914:7-16

microfluidic PCR examples

(a) The channels are filled with different dyes for visualization.

(b) Placement of the probes for the different genes.

Ahrberg et al, Lab Chip , 2016, 16, 3866

85bp DNA are amplified in 17min within 34 cycles

denaturation

input output

annealing

extension

50 000 cells in a single experiment

Parallel amplification of many genes

Various designs and

approaches of

microfluidic PCR

devices

microfluidic PCR: Digital PCR (dPCR)

• partition volumes: > 5 pL

• the number of partitions: >100 000 for a single experiment

DNA undergoes limiting dilution

dPCR APPLICATIONS:

viral detection

prognostic monitoring

fetal screening

to research applications such as phage–host interactions and intracellular profiling

dPCR should be performed when

rare events must be quantified

individual mutations must be detected

ADVANTAGES of dPCR:

• No need to rely on references or standards

• Increased precision

• Capability to analyze complex mixtures

• Linear detection of small-fold changes

microfluidic PCR: Digital PCR (dPCR)

dPCR data output

Fig. 2. Examples of data

output from a droplet dPCR

instrument (Bio-Rad

QX100). Clear distinction

between positive and

negative partitions (A and

B); no-template control

reactions (C and D), and

temperature optimization

showing incremental

separation between positive

and negative partitions

from wells C02 to G02 (E

and F).

2 targets

positive

negative

no-template

control

negative

no-t

emp

late

con

trol

neg

ati

ve

posi

tive

neg

ati

ve

posi

tive

neg

ati

ve

Amplitude

AmplitudeAmplitude

Events

Events

positive

negative

Flu

ore

scen

ce

Flu

ore

scen

ce

Flu

ore

scen

ce

Fre

qu

ency

Fre

qu

ency

Fre

qu

ency

Events

qRT-PCR reactions: One-step vs Two-step: limiting sample amount?

R packages:

SLqPCR, EasyqpcR, ddCt, NormqPCR, ReadqPCR, FPK-PCR, HTqPCR

for housekeeping genes:

geNorm, NormFinder, BestFinder, BestKeeper, Genenvestigator, RefGenes, REST

PCR algorithms, packages…

Science. 1991 Jun 21;252(5013):1643-51.Recent advances in the polymerase chain reaction.Erlich HA1, Gelfand D, Sninsky JJ.

Lab Chip. 2016 Oct 5;16(20):3866-3884.Polymerase chain reaction in microfluidic devices.Ahrberg CD1, Manz A2, Chung BG1

Methods Mol Biol. 2014;1169:133-42. doi: 10.1007/978-1-4939-0882-0_13.Expression profiling by real-time quantitative polymerase chain reaction (RT-qPCR).Lech M1, Anders HJ.

Expert Rev Mol Diagn. 2005 Mar;5(2):209-19.Basic principles of real-time quantitative PCR.Arya M1, Shergill IS, Williamson M, Gommersall L, Arya N, Patel HR.

Nat Protoc. 2006;1(3):1559-82.Quantification of mRNA using real-time RT-PCR.Nolan T1, Hands RE, Bustin SA.

Anal Biochem. 2005 Sep 1;344(1):141-3.The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization.Dheda K1, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GA, Zumla A.

Methods. 2001 Dec;25(4):402-8.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Livak KJ1, Schmittgen TD.

Nucleic Acids Res. 2001 May 1;29(9):e45.A new mathematical model for relative quantification in real-time RT-PCR.Pfaffl MW1.

For extra info

J Vis Exp. 2012 May 22;(63):e3998. doi: 10.3791/3998.Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.Lorenz TC1.

Sci Rep. 2017 Aug 29;7(1):9617. doi: 10.1038/s41598-017-09183-4.Poisson Plus Quantification for Digital PCR Systems.Majumdar N1, Banerjee S2, Pallas M2, Wessel T2, Hegerich P2.

Clin Chem. 2013 Jun;59(6):892-902. doi: 10.1373/clinchem.2013.206375. Epub 2013 Apr 9.The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments.Huggett JF1, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Hellemans J, Kubista M, Mueller RD, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT, Bustin SA.

Clin Chem. 2015 Jan;61(1):79-88. doi: 10.1373/clinchem.2014.221366. Epub 2014 Oct 22.Considerations for digital PCR as an accurate molecular diagnostic tool.Huggett JF1, Cowen S2, Foy CA2.

Anal Chem. 2011 Nov 15;83(22):8604-10. doi: 10.1021/ac202028g. Epub 2011 Oct 28.High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.Hindson BJ1, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW, Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, ErndtNG, Cauley TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang S, Stumbo DP, Hodges SP, Romine S, Milanovich FP, White HE, Regan JF, Karlin-Neumann GA, Hindson CM, Saxonov S, Colston BW.

Anal Chim Acta. 2016 Mar 31;914:7-16. doi: 10.1016/j.aca.2016.02.006. Epub 2016 Feb 13.A review on continuous-flow microfluidic PCR in droplets: Advances, challenges and future.Zhang Y1, Jiang HR2.

Lab Chip. 2016 Oct 5;16(20):3866-3884.Polymerase chain reaction in microfluidic devices.Ahrberg CD1, Manz A2, Chung BG1.

For extra info

Polymerase Chain Reaction (PCR)key-notes

PCR steps: 1. denaturation, 2. annealing, 3. extension

PCR

Absolute with a standard curve

Relative with reference genes

endpoint PCRdigital PCR

microfluidic PCR

Real-time PCRvs.

Polymerase Chain Reaction (PCR)

questions?

At the bench side..