Inverse PCR can be used to amplify the sequences flanking a segment of the border sequences which are known. This is done by using prime oligonucleotides complementary to the 5‘ends of the desired segment. This orients the free 3‘OH of the primers outward of the sequence, as a result the newly synthesized chain grows away from the borders of the concerned segment.
The procedure involves-
(1) Restriction of the target DNA with a restriction enzyme that produces sticky ends and that cuts at unknown sites on either side of the known region.
(2) The restriction fragment is allowed to circularize, that is unknown region of interest.
(3) The circularized fragment is ligated and then opened with an enzyme that cuts only within the known region.
(4) The resulting linear fragment now has the known region on both its ends which can therefore be exponentially amplified using the primers specific to the known region.
RT-PCR (Reverse Transcription- PCR)
RT-PCR is used to amplify RNA sequences into DNA. RT-PCR is used to selectively amplify the desired cDNA molecule from a mixture of cDNAs. One must know the base sequence just preceding the 3‘-poly-A tail. An oligonucleotide having this base sequence is used as a gene-specific primer in the PCR. The steps involved in RT-PCR are; firstly the total mRNA is used to produce the cDNA duplex. Secondly an oligo-C primer and the gene-specific primer are used for PCR amplification of the desired cDNA present among the mixture of cDNAs. Alternatively, the gene-specific primer (complimentary to the 3‘-end of the desired mRNA) is itself used to prime reverse transcription to generate the cDNA single-strand, which is copied using a primer specific to the 5‘-end of the mRNA. The two specific primers are used to prime the PCR for amplifying the cDNA. The use of 5‘-specific primer eliminates the risk of amplification of partial cDNAs. A modification of RT-PCR is called RACE (Rapid Amplification of cDNA Ends).
Thermal Cycle Sequencing PCR
In thermal cycle sequencing PCR, only one primer is used in combination with dideoxynucleotides. The primer is either radio or fluorescence labeled. The product of PCR reaction is subjected to PAGE for determining the base sequence of the template DNA.
This type of PCR is initiated at very high annealing temperatures which allow only perfectly matched primer-template DNA hybrids to form and support amplification. The annealing temperature is dropped in a stepwise fashion with each cycle of PCR. This variation of PCR has been devised to increase the specificity of PCR without lowering the efficiency.
Hot start PCR
In hot start PCR, a critical component like Mg2+ or Taq polymerase, is left out from the reaction mixture. Once the reaction mixture is heated to the denaturation temperature, this reagent is added to the PCR tubes by opening their tops. This is done in order to check the activity of Taq polymerase which shows a degree of activity at room with amplification of spurious sequences.
Nested PCR is used in situations in which it is necessary to increase the sensitivity and/or specificity of PCR. For example, when amplifying a particular member of a polymorphic gene family or when amplifying a cDNA copy of an mRNA present at very low abundance in a clinical specimen containing several different cell types (a heterogeneous population of cells). This PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets (instead of one pair) of primers are used in two successive PCRs. In the first reaction, on pair of primers ―outer pair‖ is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments.
The product(s) are then used in a second PCR after the reaction is diluted with a set of second set ―nested or internal‖ primers whose binding sites are completely or partially different from and located 3′ of each of the primers used in the first reaction. The specificity of PCR is determined by the specificity of the PCR primers. For example, if primers bind to more than one locus (e.g. paralog or common domain), then more than one segment of DNA will be amplified. To control for these possibilities, investigators of ten employ nested primers to ensure specificity. Nested PCR means that two pairs of PCR primers were used for a single locus. The first pair amplified the locus as seen in any PCR experiment. The second pair of primers (nested primers) binds within the first PCR product and produce a second PCR product that will be shorter than the first one. The logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers.
Uses of Nested PCR
When a complete genome sequence is known, it is easier to be sure that it will not amplify the wrong locus but since very few of the world’s genomes have been sequenced completely, nested primers will continue to be an important control for many experiments.
Quantatative PCR Used to measure the quantity of a PCR product (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of precision. QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR.
Asymmetric PCR This reaction preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. A recent modification on this process, known as Linear After The Exponential-
PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.