polymerase chain reaction...•pcr reaction mixture usually includes water, buffer, magnesium...
TRANSCRIPT
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
Νουκλεϊκό οξύ, DNA
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
Νουκλεϊκό οξύ, DNA
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
• 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
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
2 cycle = 4 Amplicon
3 cycle = 8 Amplicon
4 cycle = 16 Amplicon
5 cycle = 32 Amplicon
6 cycle = 64 Amplicon
7 cycle = 128 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
• 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
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
• 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
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
• 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
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
FRET
FRET
TaqMan Chemistry
TaqMan Chemistry
•
• 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
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
ELOSA
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
• 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
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