MOLECULAR AMPLIFICATION TECHNIQUES

MOLECULAR AMPLIFICATION TECHNIQUES

AND

INSTRUMENTATION
AMPLIFICATION TECHNIQUES
Polymerase Chain Reaction (PCR)
Nucleic Acid Sequence-Based Amplification (NASBA)
Strand Displacement Amplification (SDA)
Q replicase amplification
Ligase Chain Reaction (LCR)
Signal amplification (compound & branched chain probes)
Instrumentation has been developed for each of these to:
Amplify target
Detect amplification product
MOLECULAR AMPLIFICATION TECHNIQUES
In vitro Nucleic Acid amplification was first described in 1971 (Kleppe).

Synthesis of tRNA gene by primer-directed DNA repair
This was not exponential.

1983 Kary Mullis postulated the concept of the polymerase chain reaction (PCR)

Remained theoretical until 1985 when Saiki published the first application of PCR (beta-Globin)
NA amplification methods fall into 3 categories
Target amplification systems

Probe amplification systems

Signal amplification
MOLECULAR AMPLIFICATION TECHNIQUES
Each system makes use of probes to detect human pathogens

System depends on the identification of a nucleic acid sequence that is unique for the target organism

Detection of the target sequence must be a consistent marker for the whole organism

Method depends on availability of sequence data. Many research programmes are now contributing sequence information (GENBANK, EMBL)
MOLECULAR AMPLIFICATION TECHNIQUES
TARGET AMPLIFICATION METHODS
PCR - reverse transcriptase PCR
multiplex PCR
nested PCR
quantitative PCR

3SR
or
NASBA (Nucleic Acid Sequence-Based Amplification)


SDA – Strand Displacement Amplification
POLYMERASE CHAIN REACTION
PCR Amplifies a Targeted Sequence
Target Sequence
DNA Strand
Double Helix
DNA Strand
Supercoiled
DNA Strand
Virus
Fundamental to PCR are the primers
which hybridise with target DNA.
PCR Primers
Primers are synthetic strands of DNA (20 - 30 bases)
Primers are specific for target organism
Primer sequences are complementary to target
Primers
Target ds DNA
PCR Cycle - Step 1 - Denaturation Template DNA by Heat (95oC)
Target Sequence
Target Sequence
PCR Cycle - Step 2 –

Temperature is lowered (Tm ) and primers anneal to target sequences
Target Sequence
Target Sequence
Primer 1
Primer 2
5’
3’
5’
5’
3’
5’
3’
3’
PCR Cycle - Step 3 -

At 72 o C Taq DNA polymerase catalyses primer extension as complementary nucleotides are incorporated
Target Sequence
Target Sequence
Primer 1
Primer 2
5’
3’
5’
5’
3’
5’
3’
3’
Taq DNA
Polymerase
End of the 1st PCR Cycle –

Results in two copies of target sequence
Target Sequence
Target Sequence
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
Variations of PCR in the Diagnostic Lab
The most common variations of standard PCR used in the diagnostic laboratory are:

Reverse Transcriptase PCR (RT-PCR)
Nested PCR (n-PCR)
Multiplex PCR (m-PCR)
Real-time PCR
PCR amplifies DNA targets

Many viruses contain a RNA genome

Amplification requires RT-PCR
Reverse Transcriptase PCR (RT-PCR)
Reverse Transcription - Step 1 –

Primer Anneals to Target RNA Sequence
Target Sequence
Primer
5’
3’
5’
3’
Reverse Transcription - Step 2 –

rTth DNA Polymerase also has RT activity Catalyses Primer Extension by Incorporating Complementary Nucleotides
Target RNA Sequence
Primer
5’
3’
5’
3’
rTth DNA Polymerase
End of Reverse Transcription - Step 3 –

Results in Synthesis of Complementary DNA (cDNA) to the RNA Target Sequence
Target RNA Sequence
cDNA
Target RNA Sequence
cDNA
PCR Step 1 - Denaturation by Heat
PCR Step 2 - Annealing of Primer to cDNA
PCR Step 3 - rTth DNA Polymerase Catalyses Primer Extension
End of 1st PCR Cycle - Yields a Double-Stranded DNA Copy (Amplicon) of the Target Sequence
cDNA
Primer
cDNA
Primer
rTth DNA Polymerase
cDNA
Amplicon
1
3
2
4
NESTED PCR
Primer 1.2
Primer 1.1
Primer 2.2
Primer 2.2
1st Round PCR
2nd Round PCR
Amplification product
The advantages of n-PCR are:

Its increased specificity (specific binding of 2nd primer pair).

Increased sensitivity (2nd round of PCR amplification)

n-PCR is used to detect organisms present in low copy numbers

Viruses in CSF (herpes simplex, JCV)
Eye samples (adenovirus, herpes simplex)
NESTED PCR
Contamination risk
Nested PCR
Need for purity
Sensitivity and Specificity
Multiplex PCR
m-PCR is a rapid method of detecting multiple targets in a single reaction
PCR reagent mix contains multiple sets of primers. Any one of these may be amplified during the PCR

Primer sets to multiple organisms
Primer sets to multiple target genes in the same organism

Major advantage is the reduction in test processing time
Herpes virus Multiplex Primers
Exo II’
Exo III
Motif A
Motif B
Motif C
aa 750
aa 1000
215 - 315
Herpesvirus DNA polymerase gene
Reference: Heringa and Argos 1994
HSV


VZV


CMV


EBV


HHV6


HHV7
HSV


VZV


CMV


EBV


HHV6


HHV7
1st Round
2nd Round
HERPES MULTIPLEX PCR
150-250 bp
Results of PCR amplification with external and internal primers
Staphylococcus aureus Genome
APPLICATION OF m-PCR
Detection of Amplification Products
Detection of Amplification Products
Agarose Gel Detection

Solid Phase Hybridisation and
Colour Detection

Real-time Detection
Gel Detection
The presence of viral DNA in the sample is confirmed by two criteria
DNA
Ladder
Samples
1 2 3
1. A band of DNA must be present
Confirmation and Identification of PCR Products

Gel electrophoresis

Confirmatory methods
Sequencing of the amplification product
Southern blotting
Restriction fragment length polymorphism (RFLP) analysis
Colour detection of PCR Products
Colour Detection of PCR Products
L – Ladder
1 - Undiluted
2 - 1 : 4
3 - 1 : 8
4 - 1 : 16
5 - 1 : 32
6 - 1 : 64
1 2 3 4 5 6
2.0

1.5

1.0

0.5

0
C/O=0.1
Gel Detection vs Colour
Specific amplicon

Unrelated amplicon
Gel methods of amplicon detection are:
limited number of specimens
carcinogenic reagents
relatively insensitive
DNA Detection
SUMMARY
PCR is now widely used in research

“in house” diagnostic tests are becoming accepted

Nested PCR needs stringent protocol to prevent contamination

Multiplex PCR tests offer great flexibility

Instrumentation offers exciting possibilities for
the future (Quantitation)
Instrumentation in PCR
PCR was first commercialised into kit form by ROCHE MOLECULAR BIOCHEMICALS (1993)

ROCHE Instrumentation introduced in 1996

Real-time Quantitative PCR instruments became available in 1998 with ABI 7700 (TaqMan) and 2000 with ROCHE LightCycler
Commercial Kits
Roche Diagnostics
CMV

HCV (Quantitative)

COST/TEST - $35
- $120
Roche COBAS
Chlamydia/N.gonorrhoea
CMV (Quantitative)
HCV
HBV
real-time
real-time
real-time
real-time
hardware
PCR Instrumentation
ADVANTAGES
No electrophoresis
Rapid cycling times
High sample throughput
Contamination free (sealed reactions)
Sensitive (3pg or 1 genome Eq of DNA)
Broad dynamic range (101 - 1010 copies)
Reproducible (CV < 2.0 %)
Defined reaction protocols
Integrated with extraction instrumentation
PCR Instrumentation
APPLICATIONS
Rapid diagnostics
Monitoring of viral load
Detection of drug resistance
Melting curve analysis
- mutation detection
- specificity analysis