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PCR-based diagnostics for

dummies

A/Prof David Whiley QPID Laboratory, QCMRI and SASVRC, Children‟s Health Service

District, and The University of Queensland, Brisbane, Australia.

Note:

The absolute basics will be covered.

Why use PCR

for diagnostics?

Polymerase chain reaction (PCR):

Advantages -

• Very sensitive (1 copy – 10 copies of DNA)

• Very specific

• Rapid

• Can detect organisms that cannot be isolated

EG. PCR Diagnosis of Meningococcal Disease

Microscopy Culture PCR

Sensitive X X √

Rapid √ X √

Specific √ √ √

Detects non-viable

organisms √ X √

Low workload √ √ X

Easy to perform √ √ X

Cheap √ √ X

EG. PCR Diagnosis of Meningococcal Disease

EG. PCR diagnosis of sexually transmitted infections

- Chlamydia (CT) & gonorrhoea (NG)

Key advantages:

- Improved sensitivity

- Can be used on non-invasive specimens (urine and self-collected specimens)


The basic principle:

Reaction mix –

• Target DNA (from target organism)

• Oligonucleotide primers (and probes).

• DNA polymerase

• dNTPs: dATP, dCTP dTTP, dGTP

• Buffering solution, including magnesium.


The basic principle:

Thermocycling - Double-stranded DNA

95oC denaturation

55oC primer annealing

72oC primer extension


dsDNA

BUG DNA 3' 5'

5' 3'

3'

5'

5'

3'


Cycle 1; step 1 - 95oC, dsDNA is denatured


Cycle 1; step 2 - 55oC, primers anneal to each strand

F Primer R Primer

3'

5'

5'

3'

5'

5'

3'

3'

R Primer

3'

5'

5'

3'


Cycle 1; step 3 - 72oC, primer extension by DNA polymerase

5'

5'

DNA polymerase

Primer extension

3'

5'

5'

3'


End of cycle 1; two copies dsDNA

5'

5' 3'

3'

3'

5'

5'

3'

5'

5' 3'

3'


Subsequent cycles (up 55 cycles): exponential amplification


Subsequent cycles (up 55 cycles): exponential amplification

3'

5'

5'

3'

5'

5' 3'

3'

Real-time PCR

vs

Conventional PCR

Real-time PCR vs conventional PCR

Conventional PCR:


1. Nucleic acid extraction

Conventional PCR:



2. PCR amplification

Conventional PCR:




3. PCR product detection

Conventional PCR:





agarose gel

plate hybridisation

thermocycler Column extraction

Conventional PCR:





Real-time PCR:





Combines these two steps

Real-time PCR:



2. PCR amplification & detection

Real-time PCR:

Combines these two steps




Real-time PCR:




ABI7500 instrument

Column extraction

Real-time PCR:


Advantages

• decreased hands on time

• rapid result turnaround times

• high sample throughput

• closed system - reduced potential for carry-over contamination

• ideal for quantitative PCR

(Broad dynamic range; 101 - 1010 copies)

• additional information eg. melting curve analysis

Real-time PCR:

ASM News: Feb 2002

LightCycler Roche

iCycler BioRad

7700 Applied Biosystems


FluorTracker Stratagene

FluorImager Molecular Dynamics


Rotorgene Corbette

Real-time PCR instrumentation

Real-time PCR instrumentation:

LightCycler 1 (Roche)

• Uses glass capillaries (10-20l)

• Fan forced air heating and cooling

• Maximum 32 samples per run

• Approx. 60 minutes per run

• light emitting diode (LED)

LightCycler 1 Roche

Real-time PCR instrumentation:

LightCycler 1 (Roche)

• Uses glass capillaries (10-20l)

• Fan forced air heating and cooling

• Maximum 32 samples per run

• Approx. 60 minutes per run

• light emitting diode (LED)

ABI7500 (Applied Biosystems)

• Uses plastic tubes (25-100l)

• Heated block

• Maximum 96 – 384 samples per run

• Approx. 2½ hours per run

• laser

LightCycler 1 Roche

ABI7500 Applied Biosystems

Fully automated nucleic acid amplification systems…

BD Viper Gen-probe Tigris

GeneXpert

http://www.google.com.au/url?sa=i&rct=j&q=genexpert&source=images&cd=&cad=rja&docid=tgOe4Hc4WDsG4M&tbnid=YCILlW3HOL0_zM:&ved=0CAUQjRw&url=http://mytown.mercurynews.com/archives/sunnyvalesun/20030402/sv-news1.shtml&ei=K3AtUcv3JKbymAWQtoDoCg&bvm=bv.42965579,d.dGI&psig=AFQjCNEdlJx_ebDcqz3MPb96iky2ATS9Og&ust=1362018709443655

Real-time PCR

Detection chemistries

Real-time PCR: detection chemistries

Two basic types:

1. “Non-specific” intercalating compounds

eg. SYBR green I & II, LCgreen

Real-time PCR: detection chemistries

Two basic types:

1. “Non-specific” intercalating compounds

eg. SYBR green I & II, LCgreen

2. Specific probe-based chemistries

eg. Nuclease (TaqMan) Probes,

Adjacent Hybridisation Probes (HybProbes)

• Binds to double-stranded DNA

• Fluoresces „only‟ when bound to DNA

SYBR green:

Light source

SYBR green:

No PCR Product

FAM

CY5 Detection channels (ABI7500)

JOE

Light source

No PCR Product

No emission

FAM


JOE

SYBR green:

Light source

PCR Amplification has occurred

Attaches only to double stranded

DNA

FAM


JOE

SYBR green:

Light source

PCR Amplification has occurred

521nm

FAM


JOE

SYBR green:

Advantages:

• No oligonucleotide-probes required

Simple

Cheap

• Post PCR analyses. Eg. Melting curve analysis / HRM

Disadvantages

• Non-specific amplification products produce signal eg. Primer dimer

SYBR green:

Probe-based chemistries…

• Single probe system

• Donor fluorophore on 5‟ end and acceptor (or “quencher”) fluorophore on 3‟ end

of probe.

3’ 5’

TaqMan probes :

TaqMan probes :

Light source

FAM


3’ 5’

BHQ1 (quencher) FAM (reporter)

JOE

TaqMan probes :

Light source

FAM


3’ 5’


JOE

No PCR Product

TaqMan probes :

Light source

FAM



JOE

3’ 5’

PCR product

TaqMan probes :

Light source

FAM



JOE

3’ 5’

PCR product

Primer

TaqMan probes :

Light source

FAM



JOE

3’ 5’

PCR product

DNA polymerase

Primer extension

TaqMan probes :

Light source

FAM



JOE

PCR product

DNA polymerase

Primer extension

3’

TaqMan probes :

Light source

FAM


BHQ1 (quencher)

JOE

PCR product

DNA polymerase

Primer extension

3’

JOE (reporter)

TaqMan probes :

Light source

FAM


BHQ1 (quencher)

JOE

PCR product

DNA polymerase

Primer extension

3’

CY5 (reporter)

Advantages:

• Probes are more specific than SYBR green-based protocols.

• TaqMan probes can be reasonably short - only require sufficient conserved

sequence for one probe (approx 25 base pairs). even smaller target sequence using modified TaqMan probes (approx 15 bp).

• LNA bases, BHQplus, minor groove binder etc.

Disadvantages

• Probes can be expensive.

• Design of probes may be technically difficult.

• TaqMan probes cannot be used for post-PCR analyses (eg. melting curve) to

characterise PCR product.

TaqMan probes :

• Two probe system

• Donor fluorophore on 3‟ end of probe 1.

• Acceptor fluorophore on 5‟ end of probe 2.

3’ 5’ 5’ 3’

Adjacent Hybridisation Probes:


3’

5’

5’

3’

Probe 2

Probe 1

LCRed-640 (acceptor) fluorescein (donor)

Light source F1 F2

F3

Detection channels (LightCycler)

3’

5’

5’

3’

Probe 2

Probe 1

LCRed-640 (acceptor) fluorescein (donor)


Light source F1 F2

F3


PCR product

3’ 5’ 5’ 3’

Probe 2 Probe 1

fluorescein (donor) LCRed-640 (acceptor)


Light source F1 F2

F3


FRET

PCR product

3’ 5’ 5’ 3’

Probe 2 Probe 1


F1 F2

F3


Light source


Light source

FRET

PCR product

3’ 5’ 5’ 3’

Probe 2 Probe 1


F1 F3



F2

Advantages:

• Can use melting curve analysis to characterise PCR product.

Disadvantages

• Requires large conserved sequence (approx 50 base pairs) to accommodate both

probes.


Characteristics of amplification curves:

Background fluorescence:

The fluorescent “noise” produced by

negative samples.

Cycle threhold (Ct) value:

Cycle number where the

amplification curves of positive

samples cross above the background

fluorescence

Sample Ct value = 20

Background fluorescence


Log-linear phase:

Cycles during which there is

exponential production of PCR product.

Ct value



Log-linear phase

Ct value


Plateau phase:

PCR reagents are exhausted and

amplification of PCR product

„stops‟.


What can go wrong?

Potential PCR problems:

Sensitivity can be affected by:


Sensitivity can be affected by: Nucleic acid extraction -

• inefficient/failed extraction

• inhibitory substances from sample

Reaction mix failure - •Poor quality reagents, including primers & probes.

Cycling problems - •Detection channel failure.

•Failure to reach temperature.

Human error

Sequence variation •primers & probes

Competitive inhibition •Multiplex PCR

•Non-specific amplification








Human error




May lead to:

- complete failure

- differences in Ct values (delayed amplification)

- differences in overall fluorescent signal.








Human error




Specificity can be affected by; Contamination

•Carry-over of PCR products

•Sample to sample

Human error

Sequence sharing (eg. genetic exchange)








Human error




Specificity can be affected by; Contamination

•Carry-over of PCR products

•Sample to sample

Human error

Sequence sharing (eg. genetic exchange)

Highlights the importance of

appropriate: - quality control & assurance

- assay design & validation

Thank you!

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