MODULE 10 : Other Amplification Techniques
RT-PCR (Reverse Transcription PCR)
Principle: RNA template is first converted into a complementary DNA (cDNA) using a
reverse transcriptase. The cDNA is then used as a template for exponential amplification
using PCR.
- A variation of PCR commonly used in the diagnosis and quantification of RNA virus
infections and analysis of mRNA transcripts such as those produced by translocations.
- The conversion is accomplished through the action of reverse transcriptase (an enzyme
isolated from RNA viruses)
- If the starting material for a procedure is RNA, RNA may first be converted to dsDNA
(best template for amplification than ssRNA
o The enzyme RT first copies the RNA single strand into a RNA:DNA hybrid
strand and uses a hairpin formation on the end of the newly synthesized DNA
strand to prime synthesis of a complementary DNA strand, replacing the
original RNA in the hybrid. The resulting dsDNA is called cDNA (copy or
complementary DNA)
- It requires primers (oligo dT primers or random hexamers – most used to prime the
synthesis of initial DNA strand)
o Using this approach, yield of cDNA will be relatively low but highly specific
for the target of interest.
o Yield of cDNA will be higher with oligo dT primers
o The highest yield of cDNA is achieved with random hexamers or decamers (6-
or 10-based long ss oligonucleotides of random sequences
▪ Oligo dT primers is 18-base long is poly T sequences
- Used to measure RNA expression profiles, to detect rRNA, to analyze gene regions
interrupted by long introns and detect microorganisms with RNA genomes.
qPCR / Quantitative PCR / Real Time PCR
Principle: “The amount of the nucleic acid present into the sample is quantified using the
fluorescent dye or using the fluorescent labeled oligos.”
A powerful technique that allows exponential amplification of DNA sequences. Its
reaction needs a pair of primers that are complementary to the sequence of interest.
- Initially performed by adding ethidium bromide to a regular PCR
- used to detect, characterize and quantify nucleic acids for numerous applications
- qPCR machine measures the intensity of fluorescence emitted by the probe at each
cycle.
oDuring the first cycles, there is not enough fluorescence to be detected, but the
reaction rapidly produces more and more amplicons and the fluorescence
builds up
- Main advantage - the confirmation of the analytes through the melting curve analysis.
o measure and quantify how many amplicons are generated and how many non-
specific or primer-dimers are formed during the PCR reaction by doing the
melting curve analysis.
- Quantitative PCR adds two elements to the standard PCR process:
o Fluorescent dye
o Fluorometer
▪ These two elements turn qPCR from a processing step in another
procedure – to a measurement technique in its own right.
-
- Thermal cyclers meant for use with qPCR include a fluorometer to detect that
fluorescence.
o The fluorometer detects that fluorescence in real time as the thermal cycler
runs, giving readings throughout the amplification process of the PCR.
- As a result, quantitative PCR is also called real-time PCR or RT-PCR.
- qPCR has the ability to measure gene expression
Multiplex PCR
Principle: Multiplex PCR is the simultaneous detection of multiple targets in a single reaction
well, with a different pair of primers for each target.
- This technique requires two or more probes that can be distinguished from each other
and detected simultaneously.
- more than one primer pair can be added to a PCR thus multiple amplifications are
primed simultaneously.
- Useful in typing or identification analyses
- assays involve designing of large number of primers, hence it is required that the
designed primer should be of appropriate length.
o primers of short length, in the range of 18-22 bases are used
o Primers with similar Tm, preferably between 55°C-60°C are used.
- Advantages;
o More information with less sample
o Higher throughput
o Cost effective — fewer dNTPs, enzymes, and other consumables
o Time saving
o Less input material required
o More data from limited starting materials
o Increased accuracy of data normalization
o Fewer pipetting errors
Nested PCR
Principle: Nested PCR is a modification of PCR that was designed to improve sensitivity
and specificity.
- Used to increase the specificity of DNA amplification by reducing the non-specific
amplification of DNA.
- A nested PCR assay has 2 sets of primers (outer pair and inner pair) for a single locus
and two successive PCR reactions.
o The first set of primers are designed to anneal to sequences upstream from the
second set of primers and are used in an initial PCR reaction.
- Main Advantage:
o gives 100% accuracy, specificity and sensitivity.
▪ For the impossible templates where the GC content might be high or
chance of non-specific banding is higher, nested PCR offers the best
results
LAMP / Loop-mediated isothermal amplification
- LAMP is an isothermal approach to nucleic acid amplification (using a single
temperature) and does not require the thermal cycling of PCR.
- a single-tube technique for the amplification of DNA and a low-cost alternative to detect
certain diseases.
- Advantages:
o temperature requirement enables use of more portable or less expensive
instruments, or even simple incubators or water baths.
o not limited by a doubling-by-cycle amplification,
o generally produces more DNA than PCR in a more rapid incubation time
▪ It is not uncommon to see ug yields in <15 minutes.
o enzymes and conditions for LAMP provide a more robust and inhibitor-tolerant
amplification system
▪ detection of DNA or RNA species directly from a variety of crude sample
preparations.
LABORATORY:
In a separate A4-sized coupon bond, draw the procedure protocol for the following:
[Link] (1-step vs. 2-step RT-PCR)
[Link] (qPCR: 1-Step vs 2-Step)
[Link] (RT-qPCR for diagnosing COVID-19)
[Link] (tutorial video for qPCR)
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