Solid phase PCR and Suicide PCR

1. Solid phase PCR
and Suicide PCR
Rana Abdulnaser Hamood Alhakimi
Rana Alhakimi

2. Solid Phase PCR
• Amplification takes place only in solid phase
• Glass
• Latex beads.
• Supports dextran.
• Polystyrene microwells.
• Old solid surface
• Nitrocellulose.
• Nylon membranes.
• lysine coated glass slides. Rana Alhakimi

3. Solid phase primers
•The 5'-ends of the primers are modified during synthesis by
one of the following groups:
- Thiol (5'-SH)
- Amino (5'-NH2)
- Phosphate (5' -P)
- Hydroxyl (5'-OH)
- Dimethoxytrityl (5'-DMT)
- Fluorescein isothiocyanate (5'-FITC).
•The primers are covalently attached to the solid surface
specifically at their 5'-terminus.
5' modified end
3' free end
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4. Cont..
•The length of the bound primers should be
high enough to detect immobilized DNA
amplification products by hybridization assay
after solid phase PCR.
•The free 3'-end allows DNA synthesis to begin.
•The covalent bond between primer and
surface should not be affected by repeated
heating/cooling cycles during the nucleic acid
amplification procedure.
5' modified end
3' free end
target DNA
New
synthesized
DNA
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5. Solid
phase
forms
• Amplification
takes place in the
solid phase. Two
primer are grafted
onto the solid
surface.
5' modified
end
3' free end
5' modified
end
3' free end
• The forward primer is
grafted onto the solid
phase only and the
reverse primer
remains in the
solution phase.
5' modified
end
3' free end
• Two primers (forward and reverse) are
grafted onto the surface (solid phase) and
at the same time two primers (forward
and reverse) are added to the solution
phase only in small amounts.
• to reduce competition between solid
and solution primers and to generate
more amplicons in solid phase.
(1) (2) (3)
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6. Cont..
• Amplification takes place in solution and solid phase.
This combination is used to increase the number of
amplicons in the solid phase.
• Other PCR components are kept in solution phase.
• Master mix (dNTPs, Taq DNA polymerase, buffer and
MgCl2)
• Nuclease-free water
• DNA patterns
• Controls
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7. 3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
• Primers (reverse and forward) are
modified on 5' and immobilized on
solid surfac.
• The reverse primer is combined
with fluorescence to detect
amplification signals.
• Other PCR components are added
to the solution
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
• Annealing: DNA (sense and
antisense) binds to immobilized
primers
• Elongation.. taq DNA
polymerase adds
nucleotides to the DNA
template. This steo is called
interface amplification.
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
• The denaturation of target
DNA is done in solution, not on
the solid phase.
Solid phase PCR principle..Form #1
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8. 3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
• Denaturation: separates
the DNA template from the
primers.
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
• Annealing: DNA molecules that bind to
immobilized primers act as a template by
binding to other free immobilized primers.
• Elongation: additional copies are created.
• This step is called surface amplification.
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
• All amplicons bind to the solid
surface.
• In all cases, the enzymatic
extension of the primer produces
a bound amplicon.
Cont.. Solid phase PCR principle
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9. • In this form, only the forward primer is
fixed on the solid surface.
• In reverse primer in the solution phase.
• The reverse primer is used to produce
more single-stranded DNA, which can act
as a template to produce more amplicons.
• Finally, a fluorescent probe is used to
detect all amplicons using flow cytometry
or confocal microscopy or fluorescence
microscopy.
Solid phase PCR
principle..Form #2
Palanisamy, R et al., 2010
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10. Cont..
• After the amplification reaction, the solid phase was washed with PBS to
remove all molecules except the surface amplification products formed
on the solid surface by the immobilized primers.
• Finally, exponentially amplified DNAs are confirmed by fluorescence
signal by repeating the process on the solid phase.
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11. Advantages of phase solid PCR
It is used in different applications (analyte
identification, DNA sequencing and large scale single
nucleotide polymorphism analysis).
It has demonstrated ease of use and custom detection.
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12. Solid-Phase PCR disadvantages
• They designed the SP-PCR technique to avoid the
difficulties encountered in conventional PCR.
• Solid phase pcr showed low amplification efficiency
compared to conventional pcr. The amplification
efficiency of solid phase PCR has been shown to be
approximately 91% lower than the conventional PCR
method.
• Therefore, for further amplification, they amplified the
DNA in solution for two cycles before solid-phase
amplification began.
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13. Cont.. Reasons for inefficiency
• Low concentration primer.
• Inefficient extension by enzymes.
• Small amount of extracted DNA.
• Fluorescent signals are low.
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14. Cont.. Reasons for inefficiency
• If the distance between the
primers is low, this prevents the
DNA target from binding.
• Short primers (if the
primer is short, newly
synthesized DNA may
mask them).
• Incorrect binding (rather
than binding to primers,
target DNA binds to newly
synthesized DNA
(amplicon).
Chin, W. H., et al 2017
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15. To solve the inefficiency
• It increases the surface area of the solid phase and allows amplification to be
done in the solution phase.
• A new technique has been developed in which the surface is covered with a
porous gel that allows the enzymes to move freely.
• The length of the primers has been increased.
• Distance between primers on solid surface adjusted
• The solid surface is coated with suitable quencher and electrodes that are very
sensitive to preliminary signals.
• DNA extraction procedures are developed
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16. Developed new techniques to solve inefficiency
• An integrated microdevice for high-risk
HPV genotyping has been successfully
developed.
• Whole process, including nucleic acid
extraction, SP-PCR reaction and
fluorescent signal reading, was
successfully and sequentially performed
on the chip within 1 hour, which greatly
reduces analysis time compared to the
commercial method.
Zhu, C., et al 2019
Fig 1. A schematic of the integrated microfluidic chip
used for genotyping high-risk human papillomavirus
(HPV) strains from clinical samples.
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17. Cont..
• The new technique has been developed to increase the low percentage of
amplification encountered in SP-PCR as a result of the low amount of DNA
extraction.
• In this technique, DNA extraction and SP-PCR were performed on the same chip.
This chip was called a microfluidic chip.
• The sample is added to well p1.
• DNA extraction reagents are stored in valves
v1-v3 and v2 are used as a mixer.
• The extracted DNA is stored in the p2 well.
• The PCR mix is stored at p3.
• SP-PCR is performed in C.
• Amplicons are stored at p4.
• There is no contamination in this technique.
Zhu, C., et al 2019
Fig 2. A schematic of the microdevice chip: (a) Top view of the chip;
(b) The on-chip peristaltic pump consisted of three valves.
(b
)
(a)
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18. Chip demonstrated high quality in DNA extraction
• The positive control (human papillomavirus) with
negative control DNA samples were extracted on
the chip.
• The quality of the on-chip DNA extraction was then
determined using another technique, RT-PCR.
• The result showed that the quality of the nucleic
acids extracted in the integrated chip was very high.
Zhu, C., et al 2019
Fig 3. DNA was extracted on the integrated chip
from di_erent concentrations of the positive
quality control HPV58 by real-time PCR.
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19. Chip shows high specificity
• Five papillomavirus genotypes were
diluted at different concentrations to
detect the sensitivity of this chip.
• ImageJ software was then applied to
detect the fluorescence intensity of the
reaction replicates.
• The result showed that the detection limit
of the chip was 50 copies per reaction.
Zhu, C., et al 2019
Fig 4. The sensitivity of the integrated chip for the detection of the
five positive control HPV genotypes.
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20. Chip shows high specificity
• After the first sample was amplified, only the
fluorescence intensity of this sample was
detected due to the high sensitivity design of this
chip.
• Therefore, the probes are sufficiently spaced
apart to allow each sample to attach to its own
probe.
• Non-specific amplification was nearly impossible
Zhu, C., et al 2019
Fig 5. The specificity of the integrated chip for the
genotyping of HPV16, 18, 31, 33, and 58.
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21. The solid phase technique was coupled to the flourecence
resounase energy transfer technique to solve the problems
associated with amplification detection.
• Many techniques have been developed to increase the amplification detection
capability. Shin and his colleagues developed a technique combining both SP-PCR
with fluorescent agents.
• The surface of the solid is covered with cr/Au electrodes.
• The forward and fluorescent reverse primer is then immobilized on the solid
surface.
• The distance between the solid surface coated with Cr/Au electrodes (quencher)
and the flourecence agents attached to the reverse primers (fluorophore) is
minimized.
• During synthesis, the fluorophore generates the energy that is quenched by the
cr/au surface, thereby generating signals.
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22. Cont..
• The target gene in this study was the
KRAS oncogene.
• Amplification at the electrode surface can
generate signals even when free
fluorescent primers are used.
• No signal in the absence of DNA as there
is no amplification on the surface.
• This indicated that the electrode is very
sensitive for amplification formation.
Shin, Y., et al., 2014
Fig 6. Solid-phase bridge based DNA amplification. (A, upper) Fluorescence signals
from the surface DNA amplification at 1 ng/ l to 100 pg/ l of amount of the target
onthe Au surface. No signals are observed in controls including NTC (negative control)
and Hyb. (hybridization). (A, lower) Normalized fluorescence signal intensity from
thesurface DNA amplification at 1 ng/ l to 100 pg/ l of amount of the target on the Au
surface.
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23. How can it affect signal amplification
• In the same study, the result showed that the signal decreased with time.
• fluorescence signals are reduced by degradation of the fluorophore, not due to
the decrease in surface amplification efficiency at the Au surface.
Shin, Y., et al., 2014
Fig 7. Performance of the Au sensor depending on the storage conditions. (A) Detection of the fluorescence signals obtained from the
surface amplification by scanner ontime dependent-manner. (B) Signal intensity from the internal DNA by using Gel electrophoresis
depending on the storage conditions (temperature and time).
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24. Suicide PCR
• Two or more amplicons are produced from a
target to obtain the desired final specific
amplicon.
• suicid pcr is a type of nested PCR.
• Mostly, it was used to detect pathogens from
old samples.
• DNA isolated from the samples is amplified
and then sequenced.
• After sequencing, mutation and variation in
different species can be investigated.
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25. Suicide PCR
• There should be no positive control. (We do not want any other target
in our sample other than our target to avoid any further binding to
the primers and to minimize contamination).
• Negative controls should test negative.A new primer sequence that
has not been amplified before in the laboratory should be used and
the target DNA should not be sequenced before.
• A primary target must be amplified.
• A second target must be amplified and sequenced to confirm a
positive result.
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26. Suicide PCR principle
• DNA extraction
• PCR amplification
• Two PCR reactions are performed in one tube to reduce
contamination.
• Components of pcr reactions: Master mix, Nuclease-free
water, DNA target, Two or more primer sets target genes.
• One set of primers (Forward and reverse) is outside
the target, and the other is inside the target DNA.
• Pcr consists of two conditions.
• first condition denaturation, denaturation, bonding,
elongation. The second condition is denaturation,
bonding, elongation and final elongation.
• Sequencing
• Gel electrophoresis
40
cycles 40
cycles
https://bn.biobulletins.com/post/the-pcr-series-suicide-pcr
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27. Suicide PCR advantages
• Absolutely no chance of getting false positive results from field or
laboratory contaminants!
• Possibility to get highly specific amplifications on every trial.
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28. Cont..
• “Suicide” PCR was used to detect DNA from 103 skin
biopsy samples from patients with definite
rickettsiosis.
• Suicide PCR showed high specificity and sensitivity
compared to other methods such as culture and
conventional PCR.
• The overall sensitivity of suicide PCR among the
biopsy sample was 68% from patients with definite
rickettsiosis.
• Suicide PCR was 2.2 times more sensitive than
culture and 1.5 times more sensitive than normal
PCR.
• Presence of antibodies to Rickettsia sp.
• Suicide had no effect on PCR results.
Fournier, P. E., & Raoult, D., 2004
Table 1. the result from suicide pcr compared to other techniques
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29. Suicide PCR Disadvantages
• It is not always possible to design a new primer pair to target a
specific DNA fragment.
• Not every newly designed primer pair may work, and it can take a
long time to detect specific molecular signatures from any sample.
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30. Suicide PCR applications
• Suicide PCR applications include the identification of microbes in old
samples that have likely been contaminated with many other
microorganisms.
• Suicide PCR is also useful for identifying specific DNA fragments from
minimally old samples.
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31. References
• Raoult, D., Aboudharam, G., Crubézy, E., Larrouy, G., Ludes, B., & Drancourt, M. (2000). Molecular
identification by “suicide PCR” of Yersinia pestis as the agent of medieval black death. Proceedings
of the National Academy of Sciences, 97(23), 12800-12803.
• Fournier, P. E., & Raoult, D. (2004). Suicide PCR on skin biopsy specimens for diagnosis of
rickettsioses. Journal of clinical microbiology, 42(8), 3428-3434.
• Palanisamy, R., Connolly, A. R., & Trau, M. (2010). Considerations of solid-phase DNA
amplification. Bioconjugate chemistry, 21(4), 690-695.
• Zhu, C., Hu, A., Cui, J., Yang, K., Zhu, X., Liu, Y., ... & Zhu, L. (2019). A lab-on-a-chip device
integrated DNA extraction and solid phase PCR array for the genotyping of high-risk HPV in clinical
samples. Micromachines, 10(8), 537.
• Shin, Y., Kim, J., & Lee, T. Y. (2014). A solid phase-bridge based DNA amplification technique with
fluorescence signal enhancement for detection of cancer biomarkers. Sensors and Actuators B:
Chemical, 199, 220-225.
• Adessi, C., Matton, G., Ayala, G., Turcatti, G., Mermod, J. J., Mayer, P., & Kawashima, E. (2000).
Solid phase DNA amplification: characterisation of primer attachment and amplification
mechanisms. Nucleic acids research, 28(20), e87-e87.
• Chin, W. H., Sun, Y., Høgberg, J., Hung, T. Q., Wolff, A., & Bang, D. D. (2017). Solid-phase PCR for
rapid multiplex detection of Salmonella spp. at the subspecies level, with amplification efficiency
comparable to conventional PCR. Analytical and bioanalytical chemistry, 409(10), 2715-2726.
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