Hot-start PCR:
Hot-start PCR is a technique that reduces non-specific priming that occurs during the preparation of the reaction components. The technique may be performed manually by simply heating the reaction components briefly at the melting temperature (e.g., 95˚C) before adding the polymerase.
Touchdown PCR:
Touchdown PCR is a variant of PCR that reduces nonspecific primer annealing by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees above the Tm of the primers used, while at the later cycles, it is a few degrees below the primer Tm . The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.
Nested PCR:
Makes use of nested primers i.e. Two sets of primers are being used in two successive PCR reactions. In the first reaction, one pair of primers is used to generate DNA products, which act as templates for the second set of primers whose binding sites are within the intended DNA target.
It increase the specificity and reduces background due to non-specific amplification of DNA. However it requires more detailed knowledge of the target sequences.
Can RNA be PCR amplified ?
Yes, but not directly
Reverse Transcriptase PCR – RT PCR
A method to amplify cDNA copies of RNA (whole or mRNA).
A sensitive and versatile technique that can be used to,
Retrieve and clone the 5’ and 3’ termini of mRNAs
Generate large cDNA libraries from very small amounts of mRNA.
Identify mutations and polymorphisms in transcribed sequences.
Measure strength of gene expression when amount of mRNA is limited.
Steps in RT-PCR
RNA isolation
Conversion to cDNA template using reverse transcriptase (first strand synthesis) and oligo dT primers
RNA digested from the hybrid using RNaseH
cDNA amplified using DNA polymerase and DNA oligo primers (second strand synthesis)
Reverse Transcriptases:
Mesophilic enzymes encoded by Avian myeloblastosis virus (AMV) and the Moloney strain of Murine leukemia virus (Mo-MLV)
Tth DNA polymerase
Real Time PCR
PCR is an exponential process, small differences in efficiency at each cycle can lead to large differences in yield of the amplified product.
Problem occurs when we need to quantitate differences in mRNA levels
Although the traditional methods of quantitating mRNA are fairly good such as northern blotting and in situ hybridization, they do not approach the ease and speed of Real Time PCR.
Real Time PCR also has a much wider dynamic range of up to 107-fold (compared to 1000-fold in conventional RT-PCR). The dynamic range of an assay determines how much the target concentration can vary and yet still be quantified. This wide dynamic range also results in a more accurate quantitation.
PCR-ELISA
PCR-ELISAs are a capture assay for nucleic acids that mimic enzyme linked immunosorbent assays. In this assay, PCR products hybridized to an immobilized capture probe. The assay thus measures sequences internal to the PCR product and is a less expensive assay and an alternative to real time PCR. PCR ELISAs are useful for detecting and differentiating between multiple targets.
Multiplex-PCR:
The use of multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences.
By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform.
Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
Applications of PCR
a)Generation of probes
b)Generation of cDNA libraries
c)Production of DNA for sequencing
d)Analysis of mutations
e)Diagnosis of monogenic diseases (single gene disorders)
f)PCR use in Pre-implantation Genetic Diagnosis (PGD).
g)PCR in forensic science
h)Site-directed Mutagenesis
i)Comparison of gene expression
j)Rapid characterization of DNAs cloned in prokaryotic vectors
k)Cloning novel members of protein families using homology PCR
l)Detection of bacteria and viruses
