ACTIVITY NO.

3 MT (LEC)

Discuss the difference and similarities of the following in tabular form

1. Traditional PCR
2. Real time PCR
3. Reverse transcriptase PCR

Digital PCR Real-time PCR Traditional PCR

Overview Measures the Measures PCR Measures the
microreactions fraction of negative amplification as it amount of
microreactions to occurs. accumulated PCR
determine absolute product at the end of
copies. the PCR cycles.

Quantitative? Yes, the fraction of Yes, because data is No, though

microreactions negative collected during the comparing the
microreactions is fit exponential growth intensity of the
to a Poisson (log) phase of PCR amplified band on a
statistical algorithm. when the quantity of gel to standards of a
the PCR product is known concentration
directly proportional can give you 'semi-
to the amount of quantitative' results.
template nucleic
acid.

Applications  Absolute  Quantitation Amplification of DNA

quantification of gene for:
of viral load expression  Sequencing
 Absolute  Microarray  Genotyping
quantification verification  Cloning
of nucleic acid  Quality
standards control and
 Absolute assay
quantification validation
of next-gene  Pathogen
sequencing detection
 Libraries  SNP
 Rare allele genotyping
detection  Copy number
 Absolute variation
quantification  MicroRNA
of gene Analysis
expression  Viral
quantitation
 siRNA/RNAi
experiments

Summary Advantages of digital Adavantages of real- Disadvantages of

PCR: time PCR: traditional PCR:
 No need to  Increased  Poor Precision
rely on dynamic  Low
references or range of sensitivity
standards detection  Short
 Desired  No post-PCR dynamic
precision can processing range < 2 logs
be achieved  Detection is  Low
by increasing capable down resolution
total number to a 2-fold  Non-
of PCR change automated
replicates  Collects data  Size-based
 More tolerant in the discrimination
to PCR exponential only
inhibitors growth phase  Results are
 Capable of of PCR not expressed
analyzing  An increase in as numbers
complex reporter  Ethidium
mixtures fluorescent bromide for
 Unlike signal is staining is not
traditional directly very
qPCR, digital proportional quantitative
PCR provides to the  Post-PCR
a linear number of processing
response to amplicons
the number generated
of copies  The cleaved
present to probe
allow for provides a
small fold permanent
change record
differences to amplification
be detected of an
amplicon