1.

Hot Start PCR: A Solution to Nonspecific Primer Binding

 Hot start PCR helps to mitigate nonspecific primer annealing, primer
dimer formation, and often enhances product yield.

 The method involves withholding one of the critical components of PCR

like magnesium ion, the enzyme DNA polymerase, PCR primers, and
dNTPs from the reaction until the temperature in the first cycle rises
above the annealing temperature.

 Another strategy involves the use of a physical barrier like wax to

segregate key reaction components.

 Specialized reagents provide an alternative route by inhibiting the

polymerase activity at room temperature, either by the binding of an
antibody, or by covalently bound inhibitors that is required for
dissociation a high temperature activation step.

2.The Benefits of Colony PCR: A Quick and Easy Way to Screen

Transformed Colonies

 Colony PCR is a technique used for screening for positive transformed

DNA vector constructs.

 Colony samples are obtained with a sterile pipette tip or toothpick and
transferred into a PCR mix.

 For cell disruption, the PCR is either run with an extended time at 95°C
(when standard polymerase is used), or with a reduced denaturation step
at 100°C (when special recombinant DNA polymerase is used).

 It is a simpler method as steps involving genomic or plasmid DNA

isolation, restriction digestion and Southern blotting are not required at
all.

 It can also be used for screening desired recombinant clones from DNA
libraries, thus reducing the time and effort required for screening of large
number of colonies.
3.NESTED PCR;-

Unlocking the Power of Nested PCR: A Guide to Improved Sensitivity and

Specificity

 Nested PCR is a modified form of PCR designed to reduce the

contamination in products occurs due to non-specific amplification or
non-specific primer binding sites.

 In practice, "outer primers" are first used in a standard PCR procedure for
a test sample.

 Then "inner primers" are used in a second PCR reaction where the
product generated in the first PCR serves as the amplification target for
the second PCR reaction.

 In this procedure, the sensitivity of the assay is increased to multiple fold

as the product of the first reaction is re-amplified in the second reaction.

 The specificity of the assay is improved as the inner primers will only
amplify if a specific product is obtained in the first PCR reaction.

4.The Benefits of Touchdown PCR: Enhancing Specificity and Product

Formation

 Touchdown PCR offers a simple and rapid means to optimize PCR

specificity, sensitivity and yield, instead of lengthy optimizations and
primer redesigning.

 TD-PCR uses an initial annealing temperature above the estimated

melting temperature (T m ) of the primers in use, then progressively
moves down to lower, more tolerant annealing temperature as the PCR
cycle continues successively.

 Any difference in melting temperature between the correct and incorrect

annealing will result in an exponential twofold advantage per cycle.

 TD-PCR has widespread applicability in standard PCR protocols like

reverse transcriptase PCR, in the construction of cDNA libraries and in
the development of screens for detecting Single nucleotide
polymorphisms.
  TD-PCR has special application for amplification of templates that are
usually difficult to amplify, and can also be used as a standard to
enhance specificity and product formation.

5.Asymmetric PCR:-
Asymmetric PCR is a method of DNA amplification that favors the amplification
of one strand of the target DNA over the other.
This is done by using a primer with a limiting concentration or by leaving out
one of the primers.
When the limiting primer has exhausted, arithmetic increase in replication
occurs through elongation of the excess primer.
This is used to generate one DNA strand as product for use in sequencing
methods and in probing hybridization.

6.MULTIPLEX PCR :-

The Benefits of Multiplex PCR Assay

 Multiplex PCR is a technique that allows for the amplification of more

than one target sequence in a single reaction.

 The primer sets must have annealing temperatures within a narrow range
and are optimized to work properly within a single reaction.

 Multiplex PCR is useful for the detection of multiple genes in a single

reaction.

Avoiding False Positives with Internal Controls

  Multiplex PCR is a technique that allows for the determination of the
amount of a particular template in a sample by exponential amplification
and internal standards.

 False positive results are often obtained because each amplified fragment
provides an internal control for the other amplified fragments.

 Multiplex PCR can be utilized to determine the amount of a particular

template in a sample by exponential amplification and internal standards.

The Benefits of Multiplex Assays in Pathogen Identification, SNP Genotyping,

and More

 Multiplex PCR is a technique used to amplify multiple targets

simultaneously.

 False positive results can be obtained due to the amplification of internal

controls.

 Multiplex PCR is commonly used in a variety of applications such as

pathogen identification, high throughput SNP genotyping, mutation
analysis, gene deletion analysis, template quantitation, linkage analysis,
and RNA detection.

 Multiplex PCR is a technique that allows for the determination of the

amount of a particular template in a sample by exponential amplification
and internal standards. False positive results are often obtained because
each amplified fragment provides an internal control for the other
amplified fragments.

7.RT PCR:-
Unrevealing the Mystery of RT-PCR: How Reverse Transcription PCR
Works

 RT-PCR is a technique used to amplify cDNA from RNA templates.

 Reverse transcriptase enzymes from different viruses are used to produce

a DNA copy from the RNA template.
  Random primers, oligo (dT) primers, or sequence-specific primers are
used to amplify cDNA.

 Alternatively, some thermostable DNA polymerases (e.g., Tth DNA

polymerase) can be used

Manganese-Activated Reverse Transcription PCR Using Thermus

thermophilus Polymerase

 Polymerase is isolated from Thermus thermophilus that has reverse

transcriptase activity.

 This activity requires manganese (Mn 2+ ) as a cofactor for activation

instead of magnesium.

 Basic PCR follows this initial reverse transcription step for amplification
of the target sequence.

RT-PCR is a technique that is used to diagnose genetic disorders and to measure

the level of specific RNA molecules within a cell or tissue.

8.Real Time PCR:-

Unlocking the Power of qPCR: Measuring DNA Amplification in Real-Time

 Real-time or Quantitative PCR (qPCR) uses the linearity of DNA

amplification to quantify absolute or relative amounts of target sequence
in a sample.

 With the help of a fluorescent reporter, the amount of generated DNA can
be measured.

 In qPCR, DNA amplification is monitored at each cycle of PCR.

 When the DNA is getting amplified logarithmically at each cycle, the

amount of fluorescence increases over the basal level.

The thermal cycle at which the signal exceeds the fluorescence detection
threshold is known as threshold cycle.

Understanding qPCR Detection Chemistries

  q PCR uses an intercalating dye to detect the presence of specific DNA
sequences

 This method is suitable when the PCR reaction generates a specific

product, as the dye is capable of intercalating into any double stranded
DNA product.

Unlocking the Power of Molecular Beacons for Targeted PCR Amplification

 Use of a primer or short oligonucleotide specific to the target of interest

can be used for PCR amplification.

 The amount of PCR product amplified is directly proportional to the

amount of fluorescence emitted by the probe.

The Benefits of Using Scorpion Primers in PCR

 A Scorpion primer is similar to a Molecular Beacon, but it contains a

PCR primer sequence.

 When the target DNA is amplified during PCR, the beacon fragment
binds to the newly synthesized DNA, and thus, separates the fluorophore
from the quencher.

 To prevent the stem-loop structure from being copied during PCR, a

“PCR blocker” is also incorporated in the hairpin.

The Advantages of Scorpion Primers in PCR

 The intra-molecular binding between the scorpion primer and the

downstream PCR product is kinetically more favorable than that of
Molecular Beacons and TaqMan probes.

 This is due to the scorpion primer's higher affinity for the target sequence.

The Scorpion System: A Different Approach to Detection

 The Scorpion system is a DNA sequencing technology that does not

require a separate probe.

 The technology is more accurate and faster than other DNA sequencing
technologies.
 The technology is being used in clinical trials to diagnose diseases.