Polymerase Chain Reaction...•PCR reaction mixture usually includes water, buffer, magnesium...

Polymerase Chain Reaction

John G. Routsias, MD, PhD

• DNA/RNA basics

• PCR

• End-point detection of PCR

• Real-time detection of PCR

• TaqMan chemistry

• Multiplex PCR Applications of real-time PCR in Microbiology

PCR/Real-Time PCR

Νουκλεϊκό οξύ, DNA

DNA Strand

Double Helix

DNA Strand Supercoiled

DNA Strand

Chromosome


Double stranded DNA

Base pairing hydrogen bond

Hydrogen bond

Phosphodiester bond

Hydrogen Bonds Cytosine (C)

Adenine (A)

Thymine (T)

Guanine (G)

Deoxyribose

(Sugar molecule) Phosphoric Acid

(Phosphate molecule)

Cytosine (C)

Adenine (A)

Thymine (T)

Guanine (G)

• Double Helix

• DNA strands run 5´ to 3´

• Adjacent opposing strands

• Complementary base pairing

(A-T, G-C)

• A-T 2 Hydrogen bonds

• G-C 3 Hydrogen bonds


5´ to 3´ Orientation of the Sugar - Phosphate Backbone

Δομή

Κατεύθυνση της αλληλουχίας

5'- A T T G T C G C C G A A T A G T C G - 3'

3'- T A A C A G C G G C T T A T C A G C - 5'

Species Specific region

• DNA/RNA basics

• PCR





PCR/Real-Time PCR

PCR timeline

• 1953 3D structure of DNA revealed - Watson + Crick

• 1983 PCR invented (Mullis/Cetus)

• 1986 taq DNA polymerase first used

• 1991 Roche pay $300,000,000 for PCR patent

• 1993 real-time PCR (Higuchi)

• 1996 First real-time PCR machine (ABI PRISM 7700

SDS)

PCR Testing Process

SPECIMEN

Plasma/Serum

Swab

Urine

Purified

Nucleic Acid

DNA or RNA

RESULT

Quantitative

Qualitative

Genetic Predisposition

Sample Prep PCR

Αρχή της PCR

• Αποδιάταξη του DNA • Πρόσδεση του εκκινητή • Επιμήκυνση του εκκινητή

Αποδιάταξη Νουκλεϊκών οξέων

• Θέρμανση • Οξύ ή Βάση • Χημικοί παράγοντες πχ. urea, guanidine hydrochloride

Αποδιάταξη με θέρμανση

Temperature 20 40 60

Tm; melting temp.

DNA

Σταθερότητα υβριδισμού • Hydrogen bonding G C A T • Degree of complementary

• Ion concentration (stringency) high conc. -> high stability

•PCR reaction mixture usually includes water, buffer, magnesium chloride (MgCl2), forward and

reverse primers, deoxynucleotides (dNTPs), polymerase, and the sample DNA (template).

Polymerase Chain Reaction (PCR)

Master Mix Components

Mg2+

Mn2+

dCTP

dGTP

dUTP

dATP

Taq DNA Polymerase

rTth DNA Polymerase

AmpErase®

Biotinylated Primer

and

OR

and

Detection

Polymerase & dNTPs

Elongation

Annealing

Denaturation

PCR Stages

Extracted DNA

Denaturation

PCR Cycle - Step 1 – Αποδιάταξη με θέρμανση

Target Sequence

Target Sequence

PCR Cycle - Step 2 – Ο βιοτινυλιωμένος εκκινητής

προσδένεται στα άκρα της αλληλουχίας στόχου

PCR Cycle - Step 3 – Η Taq DNA Polymerase καταλύει την

επιμήκυνσή του εκκινήτη με την ενσωμάτωση

συμπληρωματικών νουκλεοτιδίων

End of the 1st PCR Cycle – Αποτέλεσμα: παραγωγή δύο

αντίγραφων της αλληλουχίας στόχου

Detection

Polymerase & dNTPs

Elongation

Annealing

Denaturation

2nd cycle

End of the 2nd PCR Cycle – Αποτέλεσμα: παραγωγή 4 αντίγραφων

της αλληλουχίας στόχου

Target Amplification

No. of No. Amplicon Cycles Copies of Target

1 2

2 4

3 8

4 16

5 32

6 64

20 1,048,576

30 1,073,741,824

1 cycle = 2 Amplicon







Παράγοντες που επηρεάζουν την PCR

• Primers (εκκινητές) • Polymerase enzyme • Co-factors MgCl2 -> DNA polymerase MnCl2 -> RNA polymerase (RT) • Temperature

• Short oligonucleotides are required to prime DNA synthesis (primers)

• Annealing of the primer to the template depends on complementary base-

pairing

• The position within the template molecule at which DNA copying is

initiated can be specified by synthesising primers with the appropriate

nucleotide sequence

Primers

Primers

• Oligonucleotide; 20-30 bp • G-C content; 50-60% • Tm. 50-80 oC • Target specificity • No primer dimer,hair-pin loop

Temperature

• Denaturation -> 95 οC • Primer annealing Tm-5 οC, Tm-10 οC • Primer extension depending on types of enzyme, eg. Taq DNA polymerase -> 72 οC

DNA polymerase

Thermus aquaticus, a thermophilic bacteria discovered in 1969 in hot spring of Yellowstone National park . It can tolerate high temperature. The DNA polymerase (Taq polymerase) was isolated.

DNA polymerase

Taq DNA polymerase • Thermus aquaticus YT1 • Temp 72 οC (75-80 οC) • Rate of nucleotide elongation 60 nucleotide/sec at 70 οC 0.25 nucleotide/sec at 25 οC • No 3’->5’ exonuclease (proof reading) • Error rate 10-5-10-6 per base

DNA polymerase

Vent DNA polymerase • Thermus litoralis • Temp 75-80 οC • Rate of nucleotide elongation 1000 nucleotides/sec • With 3’ -> 5’ exonuclease

Αξιοπιστία του πολλαπλασιασμού

• Enzyme “ 3’->5’ exonuclease ” • Optimization of reagents Mg++, conc. of nucleotide • Sequence of target dinucleotide repeat; CA repeat > slippage of polymerase -> ladder bands

Τύποι PCR

• Single PCR • Nested PCR • Multiplex PCR • Reverse transcriptase PCR • Asymmetric PCR • Quantitative PCR

Nested PCR

Primary PCR product

Nested PCR product

Target

1ry primer Nested primer

Reverse transcription PCR Amplicor HIV-1 Monitor Test Roche

1.Target RNA

2.Reverse transcription

cDNA synthesis

3.1st cycle of PCR

4.2nd cycle of PCR

Analysis of PCR

Once the amplification has taken place the PCR product (Amplicons) must now be detected.

– End Point Analysis - detection is performed on completion of the PCR

– Real-Time PCR - detection is performed after each PCR cycle

• DNA/RNA basics

• PCR





PCR/Real-Time PCR

Detection of PCR product

• Electrophoresis

DNA marker

End Point Analysis

M

M - Molecular Weight Markers

P - Positive Control

P S1 S2 S3 S4 S5 S6 N M M P N S7 S8 S9

N - Negative Control

S1 - S12 = Patient Samples

S3, S8, S9 & S12 = NEG

S10 S11 S12

Detection of PCR product

• Hybridization Dot blot Southern blot

• DNA/RNA basics

• PCR





PCR/Real-Time PCR

Real-Time Analysis

0

200000000

400000000

600000000

800000000

1000000000

1200000000

1400000000

1600000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

CYCLE NUMBER AMOUNT OF DNA0 11 22 43 84 165 326 647 1288 2569 512

10 1,02411 2,04812 4,09613 8,19214 16,38415 32,76816 65,53617 131,07218 262,14419 524,28820 1,048,57621 2,097,15222 4,194,30423 8,388,60824 16,777,21625 33,554,43226 67,108,86427 134,217,72828 268,435,45629 536,870,91230 1,073,741,82431 1,400,000,00032 1,500,000,00033 1,550,000,00034 1,580,000,000

Real-time PCR fluorescent detection methods

• Generic Detection - Intercalating and DNA binding dyes (Ethidium bromide and minor

groove binding dyes e.g. SYBR Green 1). These dyes bind to dsDNA with enhanced

fluorescence (20-100 fold increase)

• Sequence-specific probes – fluorophore-coupled nucleic acid probes. Higher

specificity. Most methods use the phenomenon of Fluorescent Resonance Energy

Transfer (FRET).

REAL TIME PCR

• kinetic approach • early stages • while still linear

www.biorad.com

Quantification with real-time PCR

SERIES OF 10-FOLD DILUTIONS

• DNA/RNA basics

• PCR





PCR/Real-Time PCR

Real-Time Analysis TaqMan Chemistry

• Double-Dye Oligonucleotide Probes

• Probes contain specific sequence relating to target of interest.

• The probe has a fluorescent label (Reporter) at the 5’ end & a fluorescent quencher at the 3’ end.

• The fluorescent label is quenched due to the FRET principle.

• 5’ exonuclease activity of Taq degrades the probe so

that the fluorescent label is separated from the

quencher (Taq multifunctional)

• When label and quencher are separated the label

fluorescent signal can be detected

Fluorescence

TaqMan Chemistry

TaqMan Chemistry

•

• DNA/RNA basics

• PCR




• Multiplex PCR

• Applications of real-time PCR in Microbiology

PCR/Real-Time PCR

Multiplexing

• Diagnostic PCR assays are usually require an internal control (duplex)

• Multiplex assays are separate targets (pathogens)

• Multiplexing more difficult than expected

• Real-time PCR can multiplex 4-6 assays maximum

• Often loose sensitivity by multiplexing (competition)

• For quantification - only duplexing probably OK

Luminex microsphere-based multiplexing system

Luminex detection

DNA microarray

• Difficult to link with DNA amplification • Difficult to manufacture arrays • Readers and software expensive

• DNA/RNA basics

• PCR




• Multiplex PCR

• Applications of real-time PCR in Microbiology

PCR/Real-Time PCR

Pathogen detection

Four main methods

1. Culture

2. Antigen detection

3. Serology

4. Molecular – Hybridization

- Nucleic Acid Amplification Tests

(NAATs) PCR etc.

PCR - not the only NA amplification method

• Other DNA amplification methods – NASBA/TMA, SDA, Loop-Amp etc.

Isothermal methods - do not require thermal cycling

• Molecular testing in diagnostic microbiology usually restricted to detection

(qualitative), quantification (viral load), and genotyping. Not usually

sequencing.

Molecular Diagnostic Testing-Viruses

• Routine viral detection

– Replace cell culture – sensitive, quick

– BBVs, Intrauterine infections, respiratory viruses, norovirus

• Viral Load Monitoring (treatment monitoring) – HIV, HCV, CMV (pre-

emptive monitoring)

• Genotyping/resistance testing – HIV, HBV, HCV

Molecular Diagnostic Testing – Bacteria/Parasites/Fungi

• Fastidious bacteria – C. trachomatis, GC, pertussis, M. genitalium, T.

whipplei, C. burnetti, B. henselae

• Rapid Bacterial diagnosis – N. meningitidis, S. pneumoniae, H. influenzae

• Antibiotic resistance – MRSA, VRE, ESBL, multi-drug resistant TB

• Parasites/Fungi – T. gondii, Malaria, P. carinii, Aspergillus

Conclusions

• PCR/real-time PCR – biggest change in technology in microbiology in last

20-25 years

• Real-time PCR implemented faster into diagnostic labs than end-point PCR

as more suitable for diagnostics

• Multiplexing remains a problem and it will be interesting to see if other

technologies supersede real-time PCR for detection/genotyping but probably

not for quantification

The end

Polymerase Chain Reaction...•PCR reaction mixture usually includes water, buffer, magnesium...

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